Neuroprotective Compositions and Methods

ABSTRACT

Neurite outgrowth-promoting prostaglandins (NEPPs) and other electrophilic compounds bind to Keap1, a negative regulator of the transcription factor Nrf2, and prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation into the nucleus of neuronal cells. Therefore, neuroprotective compositions and related methods are provided that employ such neuroprotective compounds, and prodrugs of such compounds, to cause dissociation of Nrf2 from a Keap1/Nrf2 complex.

RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. §119(e), of: U.S.Provisional Application No. 60/850,860, which was filed on Oct. 10,2006, and U.S. Provisional Application No. 60/850,918, which was filedon Oct. 10, 2006, both of which are of same title and named Stuart A.Lipton and Takumi Satoh, as inventors. The entirety of both the aforereferenced applications and documents are incorporated by reference.

STATEMENT OF GOVERNMENT RIGHTS

The invention was supported, at least in part, by a grant from theGovernment of the United States of America (grants no. P01 HD29587, R01NS43242, and R01 EY05477 from the National Institutes of Health). TheGovernment may have certain rights to the invention.

FIELD OF THE INVENTION

The present inventions relates to the field of neuroprotection andtreatment of conditions related to oxidative stress leading to neuronalinjury and death.

BACKGROUND

Cellular antioxidants are crucial for reducing oxidative stress andpreventing neuronal death. A recently elucidated pathway to induceantioxidant enzymes involves transcriptional activation through theantioxidant-responsive element (ARE) (Itoh et al., Mol. Cell. Biol.24:36-45, 2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002).In this case, electrophilic agents induce a set of genes encoding “phase2” enzymes, including hemeoxygenase-1 (HO-1), NADPH quinineoxidoreductase 1, and γ-glutamyl cysteine ligase (γ-GCL, also known asγ-glutamate cysteine ligase or γ-glutamyl cysteine ligase [γ-GCS]).These enzymes provide efficient cytoprotection, in part, by regulatingthe intracellular redox state (Itoh et al., Mol. Cell. Biol. 24:36-45,2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002).

The ARE is a cis-acting element essential for transcriptional activationof phase 2 genes by electrophiles (Itoh et al., Mol. Cell. Biol.24:36-45, 2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002).The transcription factor Nfr2 complexes with Maf family proteins totransactivate the ARE. Under basal conditions, the cytosolic regulatoryprotein Keap1 binds tightly to Nrf2, retaining it in the cytoplasm (Itohet al., Mol. Cell. Biol. 24:36-45, 2004; Gong et al., Antioxid. RedoxSignal. 4:249-257, 2002). In this regard, the action of Keap1 isanalogous to that of IκB, preventing activation and translocation of thetranscription factor NF-κB (Itoh et al., Mol. Cell. Biol. 24:36-45,2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002). In the caseof Keap1, electrophiles make a Michael adduct with critical cysteineresidues in this regulatory protein, causing the liberation of Nrf2 andallowing it to translocate into the nucleus (Itoh et al., Mol. Cell.Biol. 24:36-45, 2004; Gong et al., Antioxid. Redox Signal. 4:249-257,2002).

Among the phase 2 enzymes, HO-1 has attracted special attention becauseof its therapeutic effects against neurodegenerative diseases (Mainesand Panahian, in Hypoxia: From Genes to the Bedside, eds. Roach et al.(New York: Kluwer), 2001, pp. 249-272; Stocker et al., Science235:1043-1046, 1987).

HO-1 oxidatively cleaves heme to biliverdin, forms CO, and releases thechelated Fe²⁺ (Maines and Panahian, in Hypoxia: From Genes to theBedside, eds. Roach et al. (New York: Kluwer), 2001, pp. 249-272).Bilirubin (a reduction product of biliverdin) serves as a potent radicalscavenger (Stocker et al., Science 235:1043-1046, 1987) and protectsneuronal ells against oxidative stress at nanomolar concentrations (Doreet al., Proc. Natl. Acad. Sci. USA 96:2445-2450, 1999). Studies usinggene-knockout and transgenic mice have confirmed the biologicalsignificance of HO-1 as a cellular antioxidant (Poss and Tanegawa, Proc.Natl. Acad. Sci. USA 94:10925-10930, 1997). HO-1 has been proposed toplay an obligatory role in endogenous defense against oxidative stress,because cells from HO-1^(−/−) mice are highly susceptible to oxidativeinsults (Poss and Tanegawa, Proc. Natl. Acad. Sci. USA 94:10925-10930,1997). The significance of HO-1 in terms of drug development againstneurodegenerative diseases is based on two facts: (i) HO producesseveral antioxidative compounds, including biliverdin and bilirubin(Dore et al., Proc. Natl. Acad. Sci. USA 96:2445-2450, 1999), and (ii)the induction of HO-1 can be regulated by various compounds (Satoh etal., Eur. J. Neurosci. 17:2249-2255, 2003). Thus, it has been proposedthat an inducer of HO-1 in neurons could represent an efficientneuroprotective compound (Maines and Panahian, in Hypoxia: From Genes tothe Bedside, eds. Roach et al. (New York: Kluwer), 2001, pp. 249-272;Dore et al., Proc. Natl. Acad. Sci. USA 96:2445-2450, 1999; Satoh etal., Eur. J. Neurosci. 17:2249-2255, 2003).

SUMMARY OF THE INVENTION

We have discovered neuroprotective electrophilic compounds, and prodrugforms of such compounds, that modulate the Keap1-Nrf2 pathway, leadingto activation of the HO-1 promoter by Nrf2. Induction of HO-1 protein isknown to play an important neuroprotective role against excitotoxicityand brain ischemia.

According to one embodiment of the invention, compositions are providedthat comprise a neuroprotective amount of an electrophilic compound, ora pharmaceutically acceptable prodrug, salt or solvate thereof, whereinthe electrophilic compound causes dissociation of Nrf2 from a Keap1/Nrf2complex in a cell of a mammal, such as, for example, an neuron.According to another embodiment, the electrophilic compound binds toKeap1, causing dissociation of Nrf2 from the Keap1/Nrf2 complex.According to another embodiment, the electrophilic compound increasesexpression of a phase 2 enzyme in the cell, including but not limited toHO-1. According to another embodiment, the electrophilic compoundaccumulates in nerve cells, such as neurons or astrocytes or both.According to another embodiment, the electrophilic compound islipophilic. According to another embodiment, the electrophilic compoundis actively transported into the cell. According to another embodimentthe composition further comprises a pharmaceutically acceptable carrier.

According to another embodiment of such compositions, the electrophiliccompound causes dissociation of Nrf2 from the Keap1/Nrf2 complex withoutsubstantially reducing GSH levels in the cell.

According to another embodiment of such compositions, the electrophiliccompound is an enone, including but not limited to curcumin, or adienone compound, including but not limited to an NEPP such as, forexample, NEPP6 or NEPP11.

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is acompound of Formula I, which has a core benzene ring:

wherein:

X₁, X₂, X₃, X₄, X₅ and X₆ are each independently H, OH, alkyl or Y,provided that at least two of X₁-X₆ are OH and at least one of X₁-X₆ isY;

Y is B—C-D or C—B-D or C—B—C-D, any of which may be attached to the corebenzene ring to form a fused ring;

B is selected from the group consisting of null, carbonyl, carboxy,ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted;

C is selected from the group consisting of null, alkyl, cycloalkyl,alkenyl, cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to the core benzenering so as to form a fused ring; and

D is selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl, and etherand ester derivatives thereof; and

ether and ester derivatives thereof.

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is acompound of Formula I as described above and X₁, X₂, X₃, X₄, X₅ and X₆are each independently H, hydroxy, alkyl or Y, provided that two ofX₁-X₆ are hydroxy (para and ortho configurations of the hydroxy groupsare preferred, and the para configuration is more preferred) and one ofX₁-X₆ is Y.

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is acompound of Formula I as described above and X₁, X₂, X₃, X₄, X₅ and X₆are each independently H, OH, alkyl or Y, provided that two of X₁-X₆ arehydroxy in a para or ortho configuration and one of X₁-X₆ is Y (that is,the compounds include a p- or o-dihydroxybenzene ring structuremonosubstituted with side chain Y).

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is such acompound of Formula I wherein: X₁, X₂, X₃, X₄, X₅ and X₆ are eachindependently H, OH, alkyl or Y, provided that two of X₁-X₆ are hydroxyin a para configuration and one of X₁-X₆ is Y (that is, the compoundsinclude a p-dihydroxybenzene ring structure monosubstituted with sidechain Y) and D is selected from the group consisting of carboxy, benzoicacid, hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxy, andether and ester derivatives thereof. Preferably: Y is B—C-D; D iscarboxy or an ester derivative thereof; and B is null or carbonyl.

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is acompound of Formula II or Formula III:

Wherein X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, and X₂₀, are eachindependently H, OH, or Y, provided that at least two of X₁₁-X₂₀ are OH;Y is B—C-D or C—B-D or C—B—C-D, any of which may be attached to a ringcarbon to form a fused ring; B is selected from the group consisting ofnull, carbonyl, carboxy, ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—,any of which is optionally substituted; C is selected from the groupconsisting of null, alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl,arylalkyl, and arylalkenyl, any of which is optionally substituted, andwhich may be attached to a ring carbon so as to form a fused ring; and Dis selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl; and etherand ester derivatives thereof. According to another embodiment, saidelectrophilic compound, or pharmaceutically acceptable prodrug, salt orsolvate thereof, is such a compound of Formula II or Formula III,wherein X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, and X₂₀, are eachindependently H, OH, or Y, provided that two of X₁₁-X₁₅ are OH, or twoof X₁₆-X₁₉ are OH, or two of X₁₁-X₁₅ are OH and two of X₁₆-X₁₉ are OH.Preferably, at least one of X₁₁-X₂₀ is Y. Preferably Y is hydrophilic ornull, and more preferably hydrophilic. Preferably said compound ofFormula II or Formula III comprises an E ring comprising two OH groupsin para or ortho configuration, and more preferably in paraconfiguration. Preferably said compound of Formula II or Formula IIIcomprises a G ring comprising two OH groups in para or orthoconfiguration, and more preferably in para configuration.

According to another embodiment, said electrophilic compound, orpharmaceutically acceptable prodrug, salt or solvate thereof, is acompound of Formula IV, Formula V or Formula VI:

wherein: X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆ and X₂₇ are each independently H,OH, oxo, or Y; Y is B—C-D or C—B-D or C—B—C-D, any of which may beattached to a ring carbon to form a fused ring; B is selected from thegroup consisting of null, carbonyl, carboxy, ether, sulfanyl, amino,—NHC(O)— and —C(O)NH—, any of which is optionally substituted; C isselected from the group consisting of null, alkyl, cycloalkyl, alkenyl,cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to a ring carbon so asto form a fused ring; and D is selected from the group consisting ofnull, carboxy, benzoic acid, hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂,NO, amino, hydroxyl; and ether and ester derivatives thereof. PreferablyB is null. Preferably said electrophilic compound, or pharmaceuticallyacceptable prodrug, salt or solvate thereof, is a compound of Formula IVor Formula V, more preferably a compound of Formula IV. Preferably X₂₁and X₂₄ are each independently methyl, carboxy, —C(O)OCH₃, CH₂OH, orCH₂OC(O)CH₃. Preferably X₂₂ and X₂₃ are each independently H, OH, oxo,or —OCH₃, and more preferably at least one of X₂₂ and X₂₃ is OH or oxo.Preferably X₂₅ or X₂₇ or both are methyl. Preferably at least one ofX₂₁, X₂₆ or X₂₇ is carboxy, and more preferably, one of X₂₁ or X₂₄ isCH₃ and the other is selected from the group consisting of carboxy,—C(O)OCH₃, —CH₂OH, and —CH₂OC(O)CH₃. The R enantiomer of X₂₆ ispreferred.

According to another embodiment of such compositions, said electrophiliccompound, or pharmaceutically acceptable prodrug, salt or solvatethereof, is selected from the group consisting of TBHQ; an NEPP; paraL-dopa; Benzeneacetic acid, 2,5-dihydroxy-α-octylidene-, (Z)-(9CI);Benzenedecanoic acid, 2,5-dihydroxy-t-oxo-(9CI); Benzeneundecanoic acid,2,5-dihydroxy-(9CI); Benzenebutanoic acid,2,5-dihydroxy-γ-oxo-β-phenyl-(9CI); Benzenebutanoic acid,2,5-dihydroxy-β-(4-methylphenyl)-γ-oxo-(9CI); Benzoic acid,2-[2-(2,5-dihydroxyphenyl)ethyl]-6-hydroxy-(9CI); Benzoic acid,5-[2-(2,5-dihydroxyphenyl)ethyl]-2-hydroxy-(9CI); Benzoic acid,4-[2-(2,5-dihydroxyphenyl)-2-oxoethyl]-(9CI); Benzoic acid,3-[[(2,5-dihydroxyphenyl)methyl]amino]-(9CI);[1,1′-Biphenyl]-4-carboxylic acid, 2′,5′-dihydroxy-(9CI); Pentanoicacid, 5-(2,5-dihydroxyphenoxy)-2,2-dimethyl-(9CI); Octanoic acid,8-[(2,5-dihydroxybenzoyl)amino]-(9CI); Benzenebutanoic acid,2,5-dihydroxy-γ-phenyl-(9CI); 5,9-Undecadienoic acid,2-[2-(2,5-dihydroxyphenyl)ethylidene]-11-hydroxy-6,10-dimethyl-,(2Z,5E,9E)-(9CI); 5,9-Undecadienoic acid,2-[2-(2,5-dihydroxyphenyl)ethylidene]-6,10-dimethyl (2Z,5E)-(9CI);Benzenebutanoic acid,α-[(3E)-4,8-dimethyl-3,7-nonadienyl]-2,5-dihydroxy-γ-oxo-, (+)-(9CI);2,6-Octadienoic acid, 8-(2,5-dihydroxyphenyl)-2,6-dimethyl-8-oxo-,(2E,6E)-(9CI); an ester of 3-(3,4)-dihydroxyphenyl]-2-propenoic acid(caffeic acid); and an ester of 3-(2,5)-dihydroxyphenyl]-2-propenoicacid.

According to another embodiment of such compositions, the compositioncomprises said prodrug of said electrophilic compound. According to oneembodiment, the prodrug is a terpenoid or flavonoid compound such as,for example, carnosic acid, para carnosic acid, or a carnosic acidderivative. According to another embodiment the cell is under oxidativestress and said prodrug is oxidized in the cell to produce theelectrophilic compound.

According to another embodiment of such compositions, the compositioncomprises an amount of said electrophilic compound, prodrug, salt orsolvate thereof that is effective for treating a member of the groupconsisting of a neurological disorder, an ophthalmological disorder, anda combination thereof in a mammal, including, without limitation, ahuman. According to another embodiment the neurological disorder, anophthalmological disorder, or a combination thereof results from atleast one member of the group consisting of trauma, ischemia, andhypoxia. According to another embodiment the neurological disorder,ophthalmological disorder, or combination thereof is selected from thegroup consisting of painful neuropathy, neuropathic pain, diabeticneuropathy, drug dependence, drug addition, drug withdrawal, nicotinewithdrawal, opiate tolerance, opiate withdrawal, depression, anxiety, amovement disorder, tardive dyskinesia, a cerebral infection thatdisrupts the blood-brain barrier, meningitis, meningoencephalitis,stroke, hypoglycemia, cardiac arrest, spinal cord trauma, head trauma,perinatal hypoxia, cardiac arrest, hypoglycemic neuronal damage,glaucoma, retinal ischemia, ischemic optic neuropathy, maculardegeneration, multiple sclerosis, sequalae of hyperhomocystinemia,convulsion, pain, schizophrenia, muscle spasm, migraine headache,urinary incontinence, emesis, brain edema, tardive dyskinesia,AIDS-induced dementia, ocular damage, retinopathy, a cognitive disorder,and a neuronal injury associated with HIV infection. According toanother embodiment the neurological disorder, ophthalmological disorder,or combination thereof is selected from the group consisting ofepilepsy, Alzheimer's disease, vascular (multi-infarct) dementia,Huntington's disease, Parkinsonism, multiple sclerosis, amyotrophiclateral sclerosis, and minimal cognitive impairment (MCI).

According to another embodiment of such compositions, the compositioncomprises an amount of such an electrophilic compound, prodrug, salt orsolvate thereof that is effective for reducing or slowing aging or asymptom thereof in a mammal.

According to another embodiment of such compositions, the compositioncomprises a neuroprotective amount of an electrophilic compound, or apharmaceutically acceptable prodrug, salt or solvate thereof, whereinthe electrophilic compound binds to Keap1 in a cell of a mammal, causingdissociation of Nrf2 from the Keap1/Nrf2 complex and increasingexpression of a phase 2 enzyme in the cell without substantiallyreducing GSH levels in the cell.

According to another embodiment of the invention, methods are providedfor preventing or delaying injury, damage or death of a cell of a mammal(due to processes including but not limited to apoptosis, necrosis orautophagy) comprising administering to the cell any of the compositionsdescribed above. According to one such embodiment, the method comprisesadministering the composition to the cell in vitro. According to onesuch embodiment, the method comprises administering the composition tothe cell in vivo, i.e., administering the composition to the mammal.

According to another embodiment of the invention, methods are providedfor treating a neurological disorder, an ophthalmological disorder, or acombination thereof in a mammal in need of such treatment, such methodscomprising administering to the mammal any of the compositions describedabove.

According to another embodiment of the invention, compounds areprovided, or pharmaceutically acceptable prodrugs, salts or solvatesthereof, of Formula IV:

wherein: X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆ and X₂₇ are each independently H,OH, oxo, or Y; Y is B—C-D or C—B-D or C—B—C-D, any of which may beattached to a ring carbon to form a fused ring; B is selected from thegroup consisting of null, carbonyl, carboxy, ether, sulfanyl, amino,—NHC(O)— and —C(O)NH—, any of which is optionally substituted; C isselected from the group consisting of null, alkyl, cycloalkyl, alkenyl,cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to a ring carbon so asto form a fused ring; and D is selected from the group consisting ofnull, carboxy, benzoic acid, hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂,NO, amino, hydroxyl; and ether and ester derivatives thereof. PreferablyB is null. Preferably X₂₁ and X₂₄ are each independently methyl,carboxy, —C(O)OCH₃, CH₂OH, or CH₂OC(O)CH₃. Preferably X₂₂ and X₂₃ areeach independently H, OH, oxo, or —OCH₃, and more preferably at leastone of X₂₂ and X₂₃ is OH or oxo. Preferably X₂₅ or X₂₇ or both aremethyl. Preferably at least one of X₂₁, X₂₆ or X₂₇ is carboxy, and morepreferably, one of X₂₁ or X₂₄ is CH₃ and the other is selected from thegroup consisting of carboxy, —C(O)OCH₃, —CH₂OH, and —CH₂OC(O)CH₃. The Renantiomer of X₂₆ is preferred.

According to another embodiment of the invention, pharmaceuticalcompositions are provided comprising: (a) a neuroprotective amount of acompound selected from member of the group consisting a compound ofFormula IV as described above, para carnosic acid, para L-dopa, paracaffeic acid or a pharmaceutically acceptable prodrug, salt or solvatethereof; and (b) a pharmaceutically acceptable carrier.

According to another embodiment of the invention, methods are providedfor identifying a neuroprotective electrophilic compound, or apharmaceutically acceptable prodrug, salt or solvate thereof, suchmethods comprising administering to a cell (in vitro or in vivo) acomposition comprising said electrophilic compound, prodrug, salt orsolvate thereof and determining whether administration of thecomposition causes dissociation of Nrf2 from the Keap1/Nrf2 complex inthe cell. According to one embodiment, such methods comprise determiningwhether the electrophilic compound binds to Keap1. According to anotherembodiment, such methods comprise determining whether administration ofthe composition causes transcriptional activation by Nrf2 in the cell.According to another embodiment, such methods comprise determiningwhether administration of the composition causes an increase inexpression of a phase 2 enzyme, including but not limited to HO-1, inthe cell. According to another embodiment, such methods comprisedetermining whether administration of the composition decreases ordelays stress-induced death of the cell (due, for example, to apoptosis,necrosis or autophagy). According to another embodiment, such methodscomprise determining whether administration of the composition protectsa test animal comprising the cell against cerebral ischemia/reperfusioninjury in a suitable in vivo assay. According to another embodiment,such methods comprise determining whether the electrophilic compoundaccumulates in nerve cells such as, for example, neurons or astrocytesor both. According to another embodiment, such methods comprisedetermining whether administration of the composition causesdissociation of Nrf2 from the Keap1/Nrf2 complex without substantiallyreducing GSH levels in the cell.

According to another embodiment of the invention, an electrophiliccompound that causes dissociation of Nrf2 from a Keap1/Nrf2 complex in acell of a patient, or a pharmaceutically acceptable prodrug, salt orsolvate thereof, is used to prepare a medicament to treat a neurologicaldisorder, an ophthalmological disorder, or a combination thereof.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description, accompanying drawings,and the claims.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows various selected neuroprotective electrophilic orproelectrophilic compounds: para L-dopa, TBHQ, NEPP 6, NEPP11, curcumin,carnosic acid and para carnosic acid.

FIG. 2 shows core chemical structures of neuroprotective electrophiliccompounds of the catechol and enone types.

FIG. 3 shows the structure of carnosic acid and various neuroprotectivecarnosic acid derivatives.

FIG. 4 shows that CA protects PC12h cells via Nrf2. (a) Nrf2DN increasescell death. PC12h cells, PC12hW1B cells, and PC12hD5D cells (expressingNrf2DN) were incubated with various concentrations of glutamate for 20h. Cell survival was assessed using the MTT assay. *Significantlydifferent (p<0.01) from PC12h cells by ANOVA. (b) Dose-dependentactivation of the ARE by CA and sulforaphane. PC12h cells weretransfected with an ARE-luciferase reporter gene plasmid, and thenincubated for 20 h with various concentrations of CA or sulforaphane.*Significantly different (p<0.01) between CA and sulforaphane by ANOVA.(c) CA protects PC12h cells in an Nrf2-dependent manner. Variousconcentrations of CA were added to PC12, PC or PC12hD5D cells 1 h priorto exposure to 5 mM glutamate for 20 h. Viability was then assessed bythe MTT assay. *Significantly different (p<0.01) from PC12h cells byANOVA.

FIG. 5 shows that CA activates the ARE and protects cortical neurons inan Nrf2-dependent manner. Cortical cultures (E17, DIV21) weretransfected with ARE-luciferase reporter gene DNA (1 μg/well) andco-transfected with pEF6 or pEFNrf2DN. CA (3 μM) or vehicle was thenadded to the cultures. After a 24-h incubation, cell lysates were usedfor luciferase reporter gene assays. Values are mean±SEM; *p<0.01 byANOVA.

FIG. 6 shows CA increased reducing equivalents of GSH in the brain. Micewere fed 0.03% CA for 1 week, and their brains were then removed, lysedand subjected to GSH and GSSG measurement.

FIG. 7 shows that CA protects against cerebral ischemia induced by 2-hMCAO/24-h reperfusion and a quantification of infarct volume by TTCstaining. CA decreased infarct volume compared to vehicle-treated mice.Data represent mean±SEM (vehicle-treated, n=9; CA-treated, n=9; *p<0.05by ANOVA).

FIG. 8 shows a proposed mechanism of neuroprotective action of CA,NEPP11 and TBHQ. NEPP11 appears to protect neurons directly, TBHQ viaeffects on astrocytes, which in turn may release survival orneurotrophic factors (Ahlgren-Beckendorf et al., Glia 15:131-142, 1999;Kosaka and Yokoi, Biol. Pharm. Bull. 26:1620-1622, 2003), and CA by a“mixed” type of protection mediated by actions on both neurons andastrocytes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As described in greater detail below, compositions and related methodsare provided that comprise neuroprotective electrophiles that activatean “electrophile counterattack” by neurons that eliminates not only theelectrophiles themselves but also free radicals such as reactive oxygenspecies (ROS), thus preventing neurodegeneration. Neuroprotection bysuch electrophilic substances entails a transcription-based mechanisminvolving the Keap1/Nrf2 signaling pathway and the induction of phase 2genes, which encode enzymes that represent a coordinated response toelectrophiles that includes increasing intracellular levels ofneuroprotective substances such as γ-GCL and HO-1. Such compounds alsoprevent, reduce or delay damage due to free radical-mediated events inthe cell. According to another embodiment, such methods comprisedetermining whether administration of the composition decreases ordelays stress-induced cell damage, injury, or death (e.g., fromapoptosis, necrosis or autophagy) of the cell. Also provided arecompositions and related methods of use that comprise prodrug forms ofsuch neuroprotective electrophiles (or “pro-electrophiles”) that aremetabolized within the body of a patient to produce such electrophiliccompounds.

FIG. 1 shows examples of neuroprotective electrophilic compounds (paraL-dopa, TBHQ, NEPP6, NEPP11, and curcumin) and proelectrophiliccompounds (carnosic acid (CA), para carnosic acid, and carnosic acidderivatives).

According to one embodiment of the invention, neuroprotectiveelectrophilic compounds according to the invention are enone-typeelectrophilic compounds, which include but are not limited to enones anddienones (FIG. 2B). An example of an enone is curcumin. Examples ofdienones include cross-conjugated dienones such as neuriteoutgrowth-promoting prostaglandins (NEPPs), including, but not limitedto, NEPP 6 and NEPP11. Dienones are preferred.

According to another embodiment of the invention, neuroprotectiveelectrophilic compounds according to the invention are catechol-typeelectrophilic compounds (FIG. 2A). Examples of other catechol-typeneuroprotective electrophiles according to the invention include but arenot limited to: tert-butyl hydroquinone (TBHQ); para L-dopa; and estersof 3-(3,4)-dihydroxyphenyl]-2-propenoic acid (caffeic acid) or3-(2,5)-dihydroxyphenyl]-2-propenoic acid that permit the compound topass through cell membranes, including, but not limited to, methyl andethyl esters.

According to one embodiment of the invention, catechol-typeneuroprotective electrophilic compounds, or pharmaceutically acceptableprodrugs, salts or solvates thereof, according to the invention havestructural formulae I:

wherein:

X₁, X₂, X₃, X₄, X₅ and X₆ are each independently H, OH, alkyl or Y,provided that at least two of X₁-X₆ are OH and at least one of X₁-X₆ isY;

Y is B—C-D or C—B-D or C—B—C-D, any of which may be attached to the corebenzene ring to form a fused ring;

B is selected from the group consisting of null, carbonyl, carboxy,ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted;

C is selected from the group consisting of null, alkyl, cycloalkyl,alkenyl, cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to the core benzenering so as to form a fused ring; and

D is selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl, and etherand ester derivatives thereof.

In another embodiment, such compounds have structural formulae I,wherein: X₁, X₂, X₃, X₄, X₅ and X₆ are each independently H, OH, alkylor Y, provided that two of X₁-X₆ are OH and one of X₁-X₆ is Y (that is,the compounds have a para, ortho or meta dihydroxybenzene ring structuremonosubstituted with side chain Y); Y is B—C-D or C—B-D or C—B—C-D anyof which may be attached to the core benzene ring to form a fused ring;B is selected from the group consisting of null, carbonyl, carboxy,ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted; C is selected from the group consisting of null,alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, arylalkyl, andarylalkenyl, any of which is optionally substituted, and which may beattached to the dihydroxybenzene ring so as to form a fused ring; and Dis selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl, and etherand ester derivatives thereof.

In another embodiment, such compounds have structural formulae I,wherein: X₁, X₂, X₃, X₄, X₅ and X₆ are each independently H, OH, alkylor Y, provided that two of X₁-X₆ are OH in a para configuration and oneof X₁-X₆ is Y (that is, the compounds include a p-dihydroxybenzene ringstructure monosubstituted with side chain Y); Y is B—C-D or C—B-D orC—B—C-D, any of which may be attached to the core benzene ring to form afused ring; B is selected from the group consisting of null, carbonyl,carboxy, ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted; C is selected from the group consisting of null,alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, arylalkyl, andarylalkenyl, any of which is optionally substituted, and which may beattached to the core benzene ring so as to form a fused ring; and D isselected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl, and etherand ester derivatives thereof.

In another embodiment, such compounds have structural formulae I,wherein: X₁, X₂, X₃, X₄, X₅ and X₆ are each independently H, OH, alkylor Y, provided that two of X₁-X₆ are OH in a para configuration and oneof X₁-X₆ is Y (that is, the compounds include a p-dihydroxybenzene ringstructure monosubstituted with side chain Y); Y is B—C-D or C—B-D orC—B—C-D any of which may be attached to the core benzene ring to form afused ring; B is selected from the group consisting of null, carbonyl,carboxy, ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted; C is selected from the group consisting of null,alkyl, cycloalkyl, heteroalkyl, alkenyl, cycloalkenyl, aryl, arylalkyl,and arylalkenyl, any of which is optionally substituted, and which maybe attached to the dihydroxybenzene ring so as to form a fused ring; andD is selected from the group consisting of carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl, and etherand ester derivatives thereof.

Preferably Y is B—C-D.

Preferably B is null or carbonyl.

Preferably D is carboxy or an ester derivative thereof.

According to another embodiment of the invention, catechol-typeneuroprotective electrophilic compounds, or pharmaceutically acceptableprodrugs, salts or solvates thereof, according to the invention have acore flavonoid structure according to structural Formulae II or FormulaIII:

wherein:

X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, and X₂₀, are eachindependently H, OH, or Y, provided that at least two of X₁₁-X₂₀ are OH;

Y is B—C-D or C—B-D or C—B—C-D, any of which may be attached to a ringcarbon to form a fused ring;

B is selected from the group consisting of null, carbonyl, carboxy,ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted;

C is selected from the group consisting of null, alkyl, cycloalkyl,alkenyl, cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to a ring carbon so asto form a fused ring; and

D is selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl;

and ether and ester derivatives thereof.

In another embodiment, such compounds have structural formulae II orIII, wherein X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, and X₂₀, areeach independently H, OH, or Y, provided that two of X₁₁-X₁₅ are OH(that is, ring E has two OH groups in para, ortho, or metaconfiguration), or two of X₁₆-X₁₉ are OH (that is, ring G has two OHgroups in para, ortho, or meta configuration) or two of X₁₁-X₁₅ are OHand two of X₁₆-X₁₉ are OH.

Preferably at least one of X₁₁-X₂₀ is Y. It is preferable that Y ishydrophilic or null, most preferably hydrophilic.

Preferably, for compounds having two OH groups on ring E, the OH groupsare in para or ortho configuration, more preferably in paraconfiguration. Similarly, it is preferable for compounds having two OHgroups on ring G that the OH groups are in para or ortho configuration,more preferably in para configuration. Most preferable are compoundshaving two OH groups in para configuration on ring E.

One example of a flavonoid compound according to the invention is thecompound below.

Other examples include, without limitation (CA Index Names):β-D-glucopyranosiduronic acid,(2S)-2-(2,5-dihydroxyphenyl)-3,4-dihydro-5-hydroxy-4-oxo-2H-1-benzopyran-7-yl;and β-D-glucopyranosiduronic acid,2-(2,5-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-1-benzopyran-7-yl.

According to another embodiment of the invention, neuroprotectivepro-electrophilic compounds, or pharmaceutically acceptable salts orsolvates thereof, according to the invention are carnosic acid (CA)derivatives having structural Formula IV, Formula V, or Formula VI:

wherein:

X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆ and X₂₇ are each independently H, OH, oxo(═O), or Y;

Y is B—C-D or C—B-D or C—B—C-D, any of which may be attached to a ringcarbon to form a fused ring;

B is selected from the group consisting of null, carbonyl, carboxy,ether, sulfanyl, amino, —NHC(O)— and —C(O)NH—, any of which isoptionally substituted;

C is selected from the group consisting of null, alkyl, cycloalkyl,alkenyl, cycloalkenyl, aryl, arylalkyl, and arylalkenyl, any of which isoptionally substituted, and which may be attached to a ring carbon so asto form a fused ring; and

D is selected from the group consisting of null, carboxy, benzoic acid,hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO, amino, hydroxyl; and etherand ester derivatives thereof.

Preferably, B is null. Compounds of Formula IV (two OH groups as shownon ring H are in para configuration) and Formula V (two OH groups asshown on ring H are in ortho configuration), are preferred, andcompounds of Formula IV are more preferred. Preferably X₂₁ and X₂₄ areeach independently methyl, carboxy, —C(O)OCH₃, CH₂OH, or CH₂OC(O)CH₃.Preferably X₂₂ and X₂₃ are each independently H, OH, oxo (═O), or —OCH₃.X₂₅ and X₂₇ are preferably methyl. Preferably, at least one of X₂₁, X₂₆or X₂₇ is carboxy, and more preferably one of X₂₁ and X₂₄ is CH₃ and theother is carboxy, —C(O)OCH₃, —CH₂OH, or —CH₂OC(O)CH₃, and at least oneof X₂₂ and X₂₃ is hydroxy or oxo. The R enantiomer of X₂₆ is preferredto the S enantiomer. The compounds of Formulae IV, V and VI should beunderstood to include, for example, carnosol and carnosol derivatives(i.e., X₂₆ is Y, wherein B and C are null and D is carboxy, and thecarboxy group is attached to the same ring carbon as X₂₂) having similarsubstituents to those described above.

FIG. 3 shows the structure of carnosic acid and various neuroprotectivecarnosic acid derivatives, showing the following changes from carnosicacid (compound 1), with reference to Formula IV or V as appropriate:compound 2, X₂₄ is carboxy; compound 3, X₂₄ is COOCH₃; compound 4, X₂₄is CH₂OH; compound 5, (ortho, Formula VI) X₂₄ is COOCH₃, (para, FormulaIV) X₂₁ is CH₂OH; compound 6, X₂₂ is oxo (═O); compound 7, X₂₂ is OH andX₂₃ is oxo (═O); and compound 8, X₂₁ is —CH₂OAc.

As used herein, the terms below have the meanings indicated.

The term “acyl,” as used herein (which may be abbreviated Ac), alone orin combination, refers to a carbonyl attached to an alkenyl, alkyl,aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety were theatom attached to the carbonyl is carbon. An “acetyl” group refers to a—C(O)CH₃ group. An “alkylcarbonyl” or “alkanoyl” group refers to analkyl group attached to the parent molecular moiety through a carbonylgroup. Examples of such groups include methylcarbonyl and ethylcarbonyl.Examples of acyl groups include formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20, preferably 2 to 6, carbonatoms. Alkenylene refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—), (—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to andincluding 20, preferably 1 to 10, and more preferably 1 to 6, carbonatoms. Alkyl groups may be optionally substituted as defined herein.Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl,octyl, noyl and the like.

The term “alkylene,” as used herein, alone or in combination, refers toa saturated aliphatic group derived from a straight or branched chainsaturated hydrocarbon attached at two or more positions, such asmethylene (—CH₂—).

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R-S-) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20, preferably from 2 to 6, morepreferably from 2 to 4, carbon atoms. “Alkynylene” refers to acarbon-carbon triple bond attached at two positions such as ethynylene(—C:::C—, —C≡C—). Examples of alkynyl radicals include ethynyl,propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl,3-methylbutyn-1-yl, hexyn-2-yl, and the like.

The terms “amido” and “carbamoyl,” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(═O)—NR₂ group with R as defined herein. The term “N-amido” as usedherein, alone or in combination, refers to a RC(═O)NH— group, with R asdefined herein. The term “acylamino” as used herein, alone or incombination, embraces an acyl group attached to the parent moietythrough an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a pendent manner or may be fused.The term “aryl” embraces aromatic radicals such as benzyl, phenyl,naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl,azulenyl, tetrahydronaphthyl, and biphenyl.

The term “arylalkenyl” or “aralkenyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkenyl group.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkynyl” or “aralkynyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl,phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C₆H₄═ derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group-with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′— group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxy,” as used herein, refers to —C(O)OH or thecorresponding “carboxylate” anion, such as is in a carboxylic acid salt.An “O-carboxy” group refers to a RC(O)O— group, where R is as definedherein. A “C-carboxy” group refers to a —C(O)OR groups where R is asdefined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” as used herein, alone or in combination, refersto a saturated or partially saturated monocyclic, bicyclic or tricyclicalkyl radical wherein each cyclic moiety contains from 3 to 12,preferably five to seven, carbon atom ring members and which mayoptionally be a benzo fused ring system which is optionally substitutedas defined herein. Examples of such cycloalkyl radicals includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and the like.“Bicyclic” and “tricyclic” as used herein are intended to include bothfused ring systems, such as decahydronapthalene, octahydronapthalene aswell as the multicyclic (multicentered) saturated or partiallyunsaturated type. The latter type of isomer is exemplified in generalby, bicyclo[1,1,1]pentane, camphor, adamantane, andbicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto 3 to 7 membered, preferably 5 to 7 membered, unsaturatedheteromonocyclic rings, or fused polycyclic rings in which at least oneof the fused rings is unsaturated, wherein at least one atom is selectedfrom the group consisting of O, S, and N. The term also embraces fusedpolycyclic groups wherein heterocyclic radicals are fused with arylradicals, wherein heteroaryl radicals are fused with other heteroarylradicals, or wherein heteroaryl radicals are fused with cycloalkylradicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl,imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl,thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl,indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl,benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl,tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl,furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclicheterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl,dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic radical containing at least one, preferably 1 to4, and more preferably 1 to 2 heteroatoms as ring members, wherein eachsaid heteroatom may be independently selected from the group consistingof nitrogen, oxygen, and sulfur, and wherein there are preferably 3 to 8ring members in each ring, more preferably 3 to 7 ring members in eachring, and most preferably 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Heterocycle groupsof the invention are exemplified by aziridinyl, azetidinyl,1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl,dihydrocinnolinyl, dihydrobenzodioxinyl,dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl,dihy-dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl,isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl,tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. Theheterocycle groups may be optionally substituted unless specificallyprohibited.

The term “hydrazinyl” as used herein, alone or in combination, refers totwo amino groups joined by a single bond, i.e., —N—N—.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The phrase “in the main chain” refers to the longest contiguous oradjacent chain of carbon atoms starting at the point of attachment of agroup to the compounds of this invention.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in combination, meanscontaining from 1 to and including 6 carbon atoms.

The term “mercaptyl” as used herein, alone or in combination, refers toan RS— group, where R is as defined herein.

The term “nitro,” as used herein, alone or in combination, refers to—NO₂.

The terms “oxy” or “oxa,” as used herein, alone or in combination, referto —O.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refer to the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocarbamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

The term “trisubstituted silyl,” as used herein, alone or incombination, refers to a silicone group substituted at its three freevalences with groups as listed herein under the definition ofsubstituted amino. Examples include trimethylsilyl,tert-butyldimethylsilyl, triphenylsilyl and the like.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that said groupis absent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, arylthio,lower alkylsulfinyl, lower alkylsulfonyl, arylsulfinyl, arylsulfonyl,arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃,C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl, thiophene, furanyl, lower carbamate,and lower urea. Two substituents may be joined together to form a fusedfive-, six-, or seven-membered carbocyclic or heterocyclic ringconsisting of zero to three heteroatoms, for example formingmethylenedioxy or ethylenedioxy. An optionally substituted group may beunsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃),monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywherein-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Wheresubstituents are recited without qualification as to substitution, bothsubstituted and unsubstituted forms are encompassed. Where a substituentis qualified as “substituted,” the substituted form is specificallyintended. Additionally, different sets of optional substituents to aparticular moiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which may be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R, R′ and R^(n) where n=(1,2, 3, . . . n), every substituent, and every term should be understoodto be independent of every other in terms of selection from a group.Should any variable, substituent, or term (e.g. aryl, heterocycle, R,etc.) occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups may be attached to a parent molecule or may occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— may beattached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds of the present invention.These centers are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and l-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds of the present invention may exist as geometric isomers. Thepresent invention includes all cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers as well as the appropriate mixtures thereof.Additionally, compounds may exist as tautomers; all tautomeric isomersare provided by this invention. Additionally, the compounds of thepresent invention can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. In general, the solvated forms are considered equivalent to theunsolvated forms for the purposes of the present invention.

Thus, for example, contemplated herein is a composition comprising the Senantiomer substantially free of the R enantiomer, or a compositioncomprising the R enantiomer substantially free of the S enantiomer. By“substantially free” it is meant that the composition comprises lessthan 10%, or less than 8%, or less than 5%, or less than 3%, or lessthan 1% of the minor enantiomer. If the particular compound comprisesmore than one chiral center, the scope of the present disclosure alsoincludes compositions comprising a mixture of the various diastereomers,as well as compositions comprising each diastereomer substantially freeof the other diastereomers. The recitation of a compound, withoutreference to any of its particular diastereomers, includes compositionscomprising all four diastereomers, compositions comprising the racemicmixture of R,R and S,S isomers, compositions comprising the racemicmixture of R,S and S,R isomers, compositions comprising the R,Renantiomer substantially free of the other diastereomers, compositionscomprising the S,S enantiomer substantially free of the otherdiastereomers, compositions comprising the R,S enantiomer substantiallyfree of the other diastereomers, and compositions comprising the S,Renantiomer substantially free of the other diastereomers.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

Additional examples of catechol-type neuroprotective electrophiles areshown in Table 1.

TABLE 1 Various catechol-type neuroprotective electrophilic compoundshaving a core benzene ring with two OH groups in para configurationCompound (CA Index Name) Structure Synthetic Compounds Benzeneaceticacid, 2,5- dihydroxy-α-octylidene-, (Z)- (9CI)

Benzenedecanoic acid, 2,5- dihydroxy-ι-oxo-(9CI) [Other names: 11-(2,5-dihydroxyphenyl) undecanoic acid; 2,5- dihydroxybenzene undecanoic acid]

Benzeneundecanoic acid, 2,5- dihydroxy-(9CI)

Benzenebutanoic acid, 2,5- dihydroxy-γ-oxo-β-phenyl- (9CI)

Benzenebutanoic acid, 2,5- dihydroxy-β-(4- methylphenyl)-γ-oxo-(9CI)

Benzoic acid, 2-[2-(2,5- dihydroxyphenyl) ethyl]-6-hydroxy-(9CI)

Benzoic acid, 5-[2-(2,5- dihydroxyphenyl) ethyl]-2-hydroxy-(9CI) [Othername: NSC 655255]

Benzoic acid, 4-[2-(2,5- dihydroxyphenyl)-2- oxoethyl]-(9CI)

Benzoic acid, 3-[[(2,5- dihydroxyphenyl) methyl]amino]-(9CI) [Othername: AG 814]

[1,1′-Biphenyl]-4-carboxylic acid, 2′,5′-dihydroxy-(9CI)

Pentanoic acid, 5-(2,5- dihydroxyphenoxy)-2,2- dimethyl-(9CI)

Octanoic acid, 8-[(2,5- dihydroxybenzoyl)amino]- (9CI)

Benzenebutanoic acid, 2,5- dihydroxy-γ-phenyl-(9CI)

Naturally Occurring Compounds 5,9-Undecadienoic acid, 2-[2- (2,5-dihydroxyphenyl)ethylidene]- 11-hydroxy-6,10-dimethyl-, (2Z,5E,9E)-(9CI)[Other name: Ganomycin A]

5,9-Undecadienoic acid, 2-[2- (2,5-dihydroxyphenyl)ethylidene]-6,10-dimethyl-, (2Z,5E)-(9CI) [Other name: Ganomycin B]

Benzenebutanoic acid, α-[(3E)- 4,8-dimethyl-3,7-nonadienyl]-2,5-dihydroxy-γ-oxo-, (+)- (9CI) [Other name: Fornicin C]

2,6-Octadienoic acid, 8-(2,5- dihydroxyphenyl)-2,6-dimethyl- 8-oxo-,(2E,6E)-(9CI) [Other Name: Orirubenone D]

Also included in the neuroprotective electrophilic compounds of theinvention are ester and ether derivatives of, for example, hydroxyl andcarboxy groups in the neuroprotective electrophilic compounds describedabove, which may increase drug delivery and chemical stability, forexample.

Examples of proelectrophiles (also referred to herein as prodrugs ofsuch neuroprotective electrophiles) according to the invention includebut are not limited to: carnosic acid and its derivatives (see FIGS. 1and 3), including, for example, para carnosic acid, carnosol, etc.Derivatives of carnosic acid are well known in the art; see, forexample, U.S. Pat. No. 6,479,549 and U.S. Patent Application No.20040014808.

Other naturally occurring and synthetic terpenoid and flavonoidcompounds are also neuroprotective.

Without intending to exclude compounds having other features, suchproelectrophiles are preferably lipophilic to enable them to passthrough the blood-brain barrier and are hydrophilic in order to be watersoluble.

Included among such proelectrophiles are compounds that can be“pathologically activated,” that is, activated by the very oxidativestress that they are meant to treat. Thus, in a target tissue underoxidative stress (such as, for example, in the brain), they areconverted to a neuroprotective electrophilic compound. The use of suchpathologically activated proelectrophiles reduces side effects sincethey are activated only, or primarily, in the injured tissue. Alsoincluded among such proelectrophiles are, for example, para analogues ofneuroprotective electrophilic compounds according to the presentinvention. Such para analogues optionally may be pathologicallyactivated).

As used herein, “neuroprotective substance” is any substance thatprotects neurons from stress, including, but not limited to, neuronalstress caused by hypoxia, ischemia, abnormal misfolded proteins,excitotoxins, free radicals, endoplasmic reticulum stressors,mitochondrial stressors (including but not limited to inhibitors of theelectron transport chain), and Golgi apparatus antagonists. Similarly,as used herein, the term “neuroprotective” refers to any detectableprotection of neurons from stress. The neuroprotective compositions andmethods of the present invention prevent or delaying cellular injury,damage or death of a cell, as is demonstrated in the Examples.Neuroprotection may be determined directly by, for example, measuringthe delay or prevention of neuronal death, such as, for example, by areduction in the number of apoptotic neurons in cerebrocortical culturesfollowing a stress. Neuroprotection may also be determined directly by,for example, measuring the severity or extent of damage to, orfunctional loss by, a tissue or organ of the nervous system followingsuch a stress, such as, for example, by measuring a decrease in the sizeof brain infarcts after MCAO/reperfusion injury. Neuroprotection may bedetermined indirectly by detecting the activation of one or morebiological mechanisms for protecting neurons, including, but not limitedto, detecting activation of the Keap1/Nrf2 pathway and/or induction ofone or more phase 2 enzymes, including but not limited tohemeoxygenase-1 (HO-1). Methods of detecting and measuring neuronalprotection are provided in the Examples below, and other such methodsare known in the art.

As used herein, “NEPP” refers to any neurite outgrowth-promotingprostaglandin (Δ⁷-prostaglandin A₁ analogues) and any derivatives andpharmaceutically acceptable salts thereof.

As used herein, “agent” refers to any substance that has a desiredbiological activity. For example, a “neuroprotective agent” hasdetectable biological activity in protecting neurons from an oxidativestress. In addition, neuroprotective agents have detectable biologicalactivity, for example, in treating conditions caused by oxidative stressand symptoms thereof, in a host, including, but not limited to, cerebralischemia/reperfusion injury (stroke) and various neurodegenerativedisorders.

As used herein, a “neurological agent” is a substance, such as achemical compound, that has an effect on the nervous system, e.g.,compounds capable of treating, inhibiting or preventing disordersaffecting the nervous system or compounds capable of eliciting aneurological and/or an ophthalmological disorder or symptoms thereof.

As used herein, “effective amount” refers to an amount of a compositionthat causes a detectable difference in an observable biological effect,including, but not limited to, a statistically significant difference insuch an effect. The detectable difference may result from a singlesubstance in the composition, from a combination of substances in thecomposition, or from the combined effects of administration of more thanone composition. For example, an “effective amount” of a neuroprotectivecomposition according to the invention, refers to an amount of thecomposition that, in a suitable in vitro or in vivo assay; detectablymeasures or otherwise indicates a delay, prevention, or reduction inneuronal death or a reduction in the severity or extent of damage to, orfunctional loss by, a tissue or organ of the nervous system following astress. Also, an “effective amount” of a neuroprotective compositionaccording to the invention refers to an amount of the composition that,in a suitable assay, detectably activates one or more biologicalmechanisms for protecting neurons, including, but not limited to,detecting activation of the Keap1/Nrf2 pathway and/or induction of oneor more phase 2 enzymes, including but not limited to hemeoxygenase-1(HO-1).

A combination of a neuroprotective substance of the present inventionand another active ingredient in a given composition or treatment may bea synergistic combination. The term “combination therapy” means theadministration of two or more therapeutic agents to treat a therapeuticcondition or disorder described in the present disclosure. Suchadministration encompasses co-administration of these therapeutic agentsin a substantially simultaneous manner, such as in a single capsulehaving a fixed ratio of active ingredients or in multiple, separatecapsules for each active ingredient. In addition, such administrationalso encompasses use of each type of therapeutic agent in a sequentialmanner. In either case, the treatment regimen will provide beneficialeffects of the drug combination in treating the conditions or disordersdescribed herein.

The term “synergy,” as described for example by Chou and Talalay, Adv.Enzyme Regul. 22:27-55 (1984), occurs when the effect of the compoundswhen administered in combination is greater than the additive effect ofthe compounds when administered alone as a single agent. In general, asynergistic effect is most clearly demonstrated at suboptimalconcentrations of the compounds. Synergy can be in terms of lowercytotoxicity, increased activity, or some other beneficial effect of thecombination compared with the individual components.

Neuroprotective substances according to the present invention canoptionally be co-administered with another neuroprotectant drug or otheractive ingredient, including, but not limited to, one or more of thefollowing: an anti-glaucoma agent, beta adrenergic blocking agent,carbonic anhydrase inhibitor, miotic agent, sympathomimetic agent,acetylcholine blocking agent, antihistamine, anti-viral agent,quinolone, anti-inflammatory agent, steroidal or non-steroidalanti-inflammatory agent, antidepressant (e.g., serotonin reuptakeinhibitor, SSRIs, etc.), psychotherapeutic agent, anti-anxiety agent,analgesic, antiseizure agent, anti-convulsant, gabapentine,anti-hypertensive agent, benzoporphyrin photosensitiser,immunosuppressive antimetabolite, anti-convulsant, barbiturate,benzodiazepine, GABA inhibitor, hydantoin, anti-psychotic, neurolaptic,antidysknetic, andrenergic agent, tricyclic antidepressant,anti-hypoglycemic, glucose solution, polypeptide hormone, antibiotic,thrombolytic agent, blood thinner, antiarrhythmic agent, corticosteroid,seizure disorder agent, anticholinesterase, dopamine blocker,antiparkinsonian agent, muscle relaxant, anxiolytic muscle relaxant, CANstimulant, antiemetic, beta adrenergic blocking agents, ergotderivative, isometheptene, antiserotonin agent, analgesic, selectiveserotonin reuptake inhibitors (SSRIs), monosamine oxidase inhibitor,AIDS adjunct agents, anti-infective agent, systemic AIDS adjunctanti-infective, AIDS chemotherapeutic agent, nucleoside reversetranscriptase, and a protease inhibitor.

As used herein, to “treat” includes (i) preventing a pathologiccondition from occurring (e.g. prophylaxis); (ii) inhibiting thepathologic condition or arresting its development; and (iii) relievingthe pathologic condition; and/or preventing or reducing the severity oneor more symptoms associated with such a pathologic condition.

As used herein, the term “patient” refers to organisms to be treated bythe compositions and methods of the present invention. Such organismsinclude, but are not limited to, mammals, including, but not limited to,humans, monkeys, dogs, cats, horses, rats, mice, etc. Such organismsalso include other organisms, and cells, tissues and organs of suchorganisms that are useful in screening for neuroprotective substancesaccording to the present invention. In the context of the invention, theterm “subject” generally refers to an individual who will receive or whohas received treatment (e.g., administration of a composition comprisinga neuroprotective substance according to the present invention).

As used herein, “pharmaceutically acceptable salts” refer to derivativesof a neuroprotective substance according to the present inventionwherein the parent compound is modified by making acid or base saltsthereof. Examples of pharmaceutically acceptable salts include, but arenot limited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, such conventional non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of a neuroprotective substanceuseful in the compositions and methods of the present invention can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,p. 1418 (1985), which is hereby incorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

The neuroprotective substance according to the present invention can beadministered as the parent compound, a pro-drug of the parent compound,or a pharmaceutically acceptable salt, solvate, or active metabolite ofthe parent compound.

“Pro-drugs” are intended to include any covalently bonded substanceswhich release the active parent drug or other formulas or compounds ofthe present invention in vivo when such pro-drug is administered to amammalian subject. Pro-drugs of a compound of the present invention areprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either in routine manipulationin vivo, to the parent compound. Examples of pro-drugs include, but arenot limited to, pro-electrophilic terpenoid or flavonoid compounds(diterpene or triterpene; for example, carnosic acid) that aremetabolized within a patient to form electrophilic compounds that areneuroprotective. As one example, such a pro-electrophilic compound couldbe activated (metabolized to an electrophilic metabolite) by oxidationin cells and tissues of the nervous system that are under oxidativestress (such as is observed in Parkinson's disease). Thus, the pro-drugwould be activated to form neuroprotective electrophilic metabolites viapathological activity, providing neuroprotection at a target site whereit is needed.

“Metabolite” refers to any substance resulting from biochemicalprocesses by which living cells interact with the active parent drug orother formulas or compounds of the present invention in vivo, when suchactive parent drug or other formulas or compounds of the present areadministered to a mammalian subject. Metabolites include products orintermediates from any metabolic pathway.

As used herein, “metabolic pathway” refers to a sequence ofenzyme-mediated reactions that transform one compound to another andprovide intermediates and energy for cellular functions. The metabolicpathway can be linear or cyclic.

As used herein, “neurological disorder” refers to any disorder of thenervous system and/or visual system. “Neurological disorders” includedisorders that involve the central nervous system (brain, brainstem andcerebellum), the peripheral nervous system (including cranial nerves),and the autonomic nervous system (parts of which are located in bothcentral and peripheral nervous system). Major groups of neurologicaldisorders include, but are not limited to, headache, stupor and coma,dementia, seizure, sleep disorders, trauma, infections, neoplasms,neuroophthalmology, movement disorders, demyelinating diseases, spinalcord disorders, and disorders of peripheral nerves, muscle andneuromuscular junctions. Addiction and mental illness, include, but arenot limited to, bipolar disorder and schizophrenia, are also included inthe definition of neurological disorder. The following is a list ofseveral neurological disorders, symptoms, signs and syndromes that canbe treated using compositions and methods according to the presentinvention: acquired epileptiform aphasia; acute disseminatedencephalomyelitis; adrenoleukodystrophy; age-related maculardegeneration; agenesis of the corpus callosum; agnosia; Aicardisyndrome; Alexander disease; Alpers' disease; alternating hemiplegia;Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis;anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia;arachnoid cysts; arachnoiditis; Anronl-Chiari malformation;arteriovenous malformation; Asperger syndrome; ataxia telegiectasia;attention deficit hyperactivity disorder; autism; autonomic dysfunction;back pain; Batten disease; Behcet's disease; Bell's palsy; benignessential blepharospasm; benign focal; amyotrophy; benign intracranialhypertension; Binswanger's disease; blepharospasm; Bloch Sulzbergersyndrome; brachial plexus injury; brain abscess; brain injury; braintumors (including glioblastoma multiforme); spinal tumor; Brown-Sequardsyndrome; Canavan disease; carpal tunnel syndrome; causalgia; centralpain syndrome; central pontine myelinolysis; cephalic disorder; cerebralaneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebralgigantism; cerebral palsy; Charcot-Marie-Tooth disease;chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyelinating polyneuropathy;chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essentialtremor; Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fronto-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy; Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactica polyneuritiformis; herpes zoster oticus; herpes zoster;Hirayama syndrome; HIV-associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly; Huntington'sdisease and other polyglutamine repeat diseases; hydranencephaly;hydrocephalus; hypercortisolism; hypoxia; immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy diseaseKinsbourne syndrome; Klippel Feil syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease—neurological sequelae; Machado-Joseph disease;macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarctdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders; multiple system atrophy with posturalhypotension; p muscular dystrophy; myasthenia gravis; myelinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus;myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome; neurological manifestations of AIDS;neurological sequelae of lupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; Parkinson's disease; paramyotonia congenital;paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome;Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy;painful neuropathy and neuropathic pain; persistent vegetative state;pervasive developmental disorders; photic sneeze reflex; phytanic acidstorage disease; Pick's disease; pinched nerve; pituitary tumors;polymyositis; porencephaly; post-polio syndrome; postherpetic neuralgia;postinfectious encephalomyelitis; postural hypotension; Prader-Willisyndrome; primary lateral sclerosis; prion diseases; progressivehemifacial atrophy; progressive multifocal leukoencephalopathy;progressive sclerosing poliodystrophy; progressive supranuclear palsy;pseudotumor cerebri; Ramsay-Hunt syndrome (types I and II); Rasmussen'sencephalitis; reflex sympathetic dystrophy syndrome; Refsum disease;repetitive motion disorders; repetitive stress injuries; restless legssyndrome; retrovirus-associated myelopathy; Rett syndrome; Reye'ssyndrome; Saint Vitus dance; Sandhoff disease; Schilder's disease;schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles;Shy-Drager syndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome;spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinalmuscular atrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome;subacute sclerosing panencephalitis; subcortical arterioscleroticencephalopathy; Sydenham chorea; syncope; syringomyelia; tardivedyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cordsyndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux;Todd's paralysis; Tourette syndrome; transient ischemic attack;transmissible spongiform encephalopathies; transverse myelitis;traumatic brain injury; tremor; trigeminal neuralgia; tropical spasticparaparesis; tuberous sclerosis; vascular dementia (multi-infarctdementia); vasculitis including temporal arteritis; Von Hippel-Lindaudisease; Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome;whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome.

As used herein, “ophthalmologic disease” or “ophthalmologic disorder”refers to disease or disorder involving the anatomy and/or function ofthe visual system, including but not limited to, glaucoma, retinalartery occlusion, ischemic optic neuropathy and macular degeneration(wet or dry).

The neurological disorder can be an affective disorder (e.g., depressionor anxiety). As used herein, “affective disorder” or “mood disorder”refers to a variety of conditions characterized by a disturbance in moodas the main feature. If mild and occasional, the feelings may be normal.If more severe, they may be a sign of a major depressive disorder ordysthymic reaction or be symptomatic of bipolar disorder. Other mooddisorders may be caused by a general medical condition. See, e.g.,Mosby's Medical, Nursing & Allied Health Dictionary, 5^(th) edition(1998).

As used herein, “depression” refers to an abnormal mood disturbancecharacterized by feelings of sadness, despair, and discouragement.Depression refers to an abnormal emotional state characterized byexaggerated feelings of sadness, melancholy, dejection, worthlessness,emptiness, and hopelessness, that are inappropriate and out ofproportion to reality. See, Mosby's Medical, Nursing & Allied HealthDictionary, 5^(th) edition (1998). Depression includes, but is notlimited to: a major depressive disorder (single episode, recurrent,mild, severe without psychotic features, severe with psychotic features,chronic, with catatonic features, with melancholic features, withatypical features, with postpartum onset, in partial remission, in fullremission), dysthymic disorder, adjustment disorder with depressed mood,adjustment disorder with mixed anxiety and depressed mood, premenstrualdysphoric disorder, minor depressive disorder, recurrent briefdepressive disorder, post-psychotic depressive disorder ofschizophrenia, a major depressive disorder associated with Parkinson'sdisease, and a major depressive disorder associated with dementia.

The neurological disorder can be pain-associated depression (PAD). Asused herein, “pain-associated depression” or “PAD” is intended to referto a depressive disorder characterized by the co-morbidity of pain andatypical depression. Specifically, the pain can be chronic pain,neuropathic pain, or a combination thereof. Specifically, the PAD caninclude atypical depression and chronic pain wherein the chronic painprecedes the atypical depression, or vice versa.

“Chronic pain” refers to pain that continues or recurs over a prolongedperiod of time (i.e., greater than three months), caused by variousdiseases or abnormal conditions, such a rheumatoid arthritis, forexample. Chronic pain may be less intense than acute pain. A person withchronic pain does not usually display increased pulse and rapidperspiration because the automatic reactions to pain cannot be sustainedfor long periods of time. Others with chronic pain may withdraw from theenvironment and concentrate solely on their affliction, totally ignoringtheir family and friends and external stimuli. See, e.g., Mosby'sMedical, Nursing & Allied Health Dictionary, 5^(th) edition (1998).

Chronic pain includes but is not limited to: lower back pain, atypicalchest pain, headache, pelvic pain, myofascial face pain, abdominal pain,and neck pain or chronic pain caused by disease or a condition such as,for example, arthritis, temporal mandibular joint dysfunction syndrome,traumatic spinal cord injury, multiple sclerosis, irritable bowelsyndrome, chronic fatigue syndrome, premenstrual syndrome, multiplechemical sensitivity, closed head injury, fibromyalgia, rheumatoidarthritis, diabetes, cancer, HIV, interstitial cystitis, migraineheadache, tension headache, post-herpetic neuralgia, peripheral nerveinjury, causalgia, post-stroke syndrome, phantom limb syndrome e, andchronic pelvic pain.

“Atypical depression” refers to a depressed affect, with the ability tofeel better temporarily in response to positive life effect (moodreactivity), plus two or more neurovegetative symptoms, including, butnot limited to: hypersomnia, increased appetite or weight gain, leadenparalysis, and a long-standing pattern of extreme sensitivity toperceived interpersonal rejection; wherein the neurovegetative symptomsare present for more than about two weeks. Such neurovegetative symptomscan be reversed compared to those found in other depressive disorders(e.g., melancholic depression).

“Acute neurological disorder” refers to a neurological disorder having arapid onset followed by a short but severe course, including, but notlimited to, febrile seizures, Guillain-Barré syndrome, stroke, andintracerebral hemorrhaging.

“Chronic neurological disorder” refers to a neurological disorderlasting for a long period of time (e.g., more than about two weeks;specifically, the chronic neurological disorder can continue or recurfor more than about four weeks, more than about eight weeks, or morethan about twelve weeks) or is marked by frequent recurrence, including,but not limited to, narcolepsy, chronic inflammatory demyelinatingpolyneuropathy, cerebral palsy, epilepsy, multiple sclerosis, dyslexia,Alzheimer's disease, and Parkinson's disease.

“Trauma” refers to any injury or shock to the body, as from violence oran accident, or to any emotional wound or shock, such as a wound orshock that causes substantial, lasting damage to the psychologicaldevelopment of a person.

“Ischemic condition” is any condition that results in a decrease in theblood supply to a bodily organ, tissue or part caused by constriction orobstruction of the blood vessels, often resulting in a reduction ofoxygen to the organ, tissue or part.

“Hypoxic conditions” are conditions in which the amount or concentrationof oxygen in the air, blood or tissue is low (subnormal).

“Painful neuropathy” or “neuropathy” is chronic pain that results fromdamage to or pathological changes of the peripheral or central nervoussystem. Peripheral neuropathic pain is also referred to as painfulneuropathy, nerve pain, sensory peripheral neuropathy, or peripheralneuritis. With neuropathy, the pain is not a symptom of injury butrather is itself the disease process. Neuropathy is not associated withthe healing process. Rather than communicating that there is an injurysomewhere, the nerves themselves malfunction and become the cause ofpain.

“Neuropathic pain” refers to pain associated with inflammation ordegeneration of the peripheral nerves, cranial nerves, spinal nerves, ora combination thereof. The pain is typically sharp, stinging, orstabbing. The underlying disorder can result in the destruction ofperipheral nerve tissue and can be accompanied by changes in skin color,temperature and edema. See, e.g., Mosby's Medical, Nursing & AlliedHealth Dictionary, 5^(th) edition (1998); and Stedman's MedicalDictionary, 25^(th) edition (1990).

“Diabetic neuropathy” refers to a peripheral nerve disorder/nerve damagecaused by diabetes, including peripheral, autonomic, and cranial nervedisorders/damage associated with diabetes. Diabetic neuropathy is acommon complication of diabetes mellitus in which nerves are damaged asa result of hyperglycemia (high blood sugar levels).

“Drug dependence” refers to habituation to, abuse of, and/or addictionto a chemical substance. Largely because of psychological craving, thelife of the drug-dependent person revolves around the need for thespecific effect of one or more chemical agents on mood or state ofconsciousness. The term thus includes not only the addiction (whichemphasizes the physiological dependence) but also drug abuse (in whichthe pathological craving for drugs seem unrelated to physicaldependence). Examples include, but are not limited to, dependence onalcohol, opiates, synthetic analgesics with morphine-like effects,barbiturates, hypnotics, sedatives, some antianxiety agents, cocaine,psychostimulants, marijuana, nicotine and psychotomimetic drugs.

“Drug withdrawal” refers to the termination of drug taking. Drugwithdrawal also refers to the clinical syndrome of psychological and,sometimes, physical factors that result from the sustained use of aparticular drug when the drug is abruptly withdrawn. Symptoms arevariable but may include anxiety, nervousness, irritability, sweating,nausea, vomiting, rapid heart rate, rapid breathing, and seizures.

“Drug addiction” or dependence is defined as having one or more of thefollowing signs: a tolerance for the drug (needing increased amounts toachieve the same effect), withdrawal symptoms, taking the drug in largeramounts than was intended or over a longer period of time than wasintended, having a persistent desire to decrease or the inability todecrease the amount of the drug consumed, spending a great deal of timeattempting to acquire the drug, or continuing to use the drug eventhough the person knows there are recurring physical or psychologicalproblems caused by the drug.

“Depression” refers to a mental state of depressed mood characterized byfeelings of sadness, despair and discouragement. Depression ranges fromnormal feelings of the blues through dysthymia to major depression.

“Anxiety disorders” refers to an excessive or inappropriate arousedstate characterized by feelings of apprehension, uncertainty, or fear.Anxiety disorders have been classified according to the severity andduration of their symptoms and specific behavioral characteristics.Categories include: generalized anxiety disorder, which is long-lastingand low-grade; panic disorder, which has more dramatic symptoms;phobias; obsessive-compulsive disorder; post-traumatic stress disorder;and separation anxiety disorder.

“Tardive dyskinesia” (e.g., Tourette's syndrome) refers to a serious,irreversible neurological disorder that can appear at any age. Tardivedyskinesia can be a side effect of long-term use ofantipsychotic/neuroleptic drugs. Symptoms can be hardly noticeable orprofound. Symptoms involve uncontrollable movement of various bodyparts, including the body, trunk, legs, arms, fingers, mouth, lips, ortongue.

“Movement disorder” refers to a group of neurological disorders thatinvolve the motor and movement systems, including, but not limited to,ataxia, Parkinson's disease, blepharospasm, Angelman syndrome, ataxiatelangiectasia, dysphonia, dystonic disorders, gait disorders,torticollis, writer's cramp, progressive supranuclear palsy,Huntington's chorea, Wilson's disease, myoclonus, spasticitiy, tardivedyskinesia, tics, Tourette syndrome, and tremors.

“Cerebral infections that disrupt the blood-brain barrier” refers toinfections of the brain or cerebrum that result in an alteration in theeffectiveness of the blood-brain barrier, either increasing ordecreasing its ability to prevent substances and/or organisms frompassing out of the bloodstream and into the central nervous system.

“Blood-brain barrier” refers to a semi-permeable layer of endothelialcells within capillaries of the central nervous system that preventslarge molecules, immune cells, many potentially damaging substances, andforeign organisms (e.g., viruses) from passing out of the bloodstreamand into the central nervous system (e.g., brain and spinal cord). Adysfunction in the blood-brain barrier may underlie in part the diseaseprocess in multiple sclerosis.

“Meningitis” refers to inflammation of the meninges of the brain andspinal cord, most often caused by a bacterial or viral infection andcharacterized by fever, vomiting, intense headache, and stiff neck.

“Meningoencephalitis” refers to inflammation of one or both of the brainand meninges.

“Stroke,” also called cerebral accident or cerebrovascular accident,refers to a sudden loss of brain function caused by a blockage orrupture of a blood vessel to the brain (resulting in a lack of oxygen tothe brain), characterized by loss of muscular control, diminution orloss of sensation or consciousness, dizziness, slurred speech, or othersymptoms that vary with the extent and severity of the damage to thebrain.

“Hypoglycemia” refers to an abnormally low level of glucose in theblood.

“Cerebral ischemia” (stroke) refers to a deficiency in blood supply tothe brain, often resulting in a lack of oxygen to the brain.

“Cardiac arrest” refers to a sudden cessation of heartbeat and cardiacfunction, resulting in a temporary or permanent loss of effectivecirculation.

“Spinal cord trauma,” also called spinal cord injury or compression,refers to damage to the spinal cord that results from direct injury tothe spinal cord itself or indirectly by damage to the bones and softtissues and vessels surrounding the spinal cord.

“Head trauma” refers to a head injury of the scalp, skull, or brain.These injuries can range from a minor bump on the skull to a devastatingbrain injury. Head trauma can be classified as either closed orpenetrating. In a closed head injury, the head sustains a blunt force bystriking against an object. A concussion is a closed head injury thatinvolves the brain. In a penetrating head injury, an object (usuallymoving at high speed, such as a windshield or other part of a motorvehicle) breaks through the skull and enters the brain.

“Perinatal hybpxia” refers to a lack of oxygen during the perinatalperiod (i.e., the period of time occurring shortly before and afterbirth, variously defined as beginning with completion of the twentiethto twenty-eighth week of gestation and ending 7 to 28 days after birth.

“Hypoglycemic neuronal damage” refers to neuronal damage, for example,nerve damage, resulting from a hypoglycemic condition (i.e., abnormallylow blood glucose levels).

“Neurodegenerative disorder” refers to a type of neurological diseasemarked by the loss of nerve cells, including, but not limited to,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,tauopathies (including fronto-temporal dementia), and Huntington'sdisease.

“Epilepsy” refers to any of various neurological disorders characterizedby sudden recurring attacks of motor, sensory, or psychic malfunctionwith or without loss of consciousness or convulsive seizures.

“Alzheimer's disease” refers to a disease marked by the loss ofcognitive ability, generally over a period of 10 to 15 years, andassociated with the development of abnormal tissues and protein deposits(plaques or tangles) in the cerebral cortex.

“Huntington's disease” refers to a hereditary disease that develops inadulthood and ends in dementia. It results from genetically programmedneuronal degeneration in certain areas of the brain that causesuncontrolled movements, loss of intellectual faculties, and emotionaldisturbance.

“Parkinsonism” refers to a disorder similar to Parkinson's disease, butwhich is caused by the effects of a medication, a differentneurodegenerative disorder, or another illness. The term “parkinsonism”also refers to any condition that causes any combination of the types ofmovement abnormalities seen in Parkinson's disease by damaging ordestroying dopamine neurons in a certain area of the brain.

“Amyotrophic lateral sclerosis” (ALS), also called Lou Gehrig's disease,refers to a progressive, fatal neurological disease. ALS belongs to aclass of disorders known as motor neuron disease. ALS occurs whenspecific nerve cells in the brain and spinal cord that control voluntarymovement gradually degenerate (usually the “upper” (i.e., in thecerebrocortex) and “lower” (in the spinal cord) motor neurons. The lossof these motor neurons causes the muscles under their control to weakenand waste away, leading to paralysis. ALS manifests itself in differentways, depending on which muscles weaken first. Symptoms may includetripping and falling, loss of motor control in hands and arms,difficulty speaking, swallowing and/or breathing, persistent fatigue,and twitching and cramping, sometimes quite severely. Upper motor neuronvariants (e.g., primary lateral sclerosis) are also included.

“Glaucoma” refers to any of a group of eye diseases characterized byabnormally high intraocular fluid pressure, damaged optic disk,hardening of the eyeball, and partial to complete loss of vision. Theretinal ganglion cells are lost in glaucoma. Some variants of glaucoma(low tension glaucoma) have normal intraocular pressure.

“Retinal ischemia” refers to a decrease in the blood supply to theretina.

“Ischemic optic neuropathy” refers to a condition that usually presentswith a sudden onset of unilaterally reduced vision. The condition is theresult of decreased blood flow to the optic nerve (ischemia). There aretwo basic types: arteritic and non-arteritic ischemic optic neuropathy.Non-arteritic ischemic optic neuropathy is generally the result ofcardiovascular disease. Patients at greatest risk have a history of highblood pressure, elevated cholesterol, smoking, diabetes, or combinationsof these. Arteritic ischemic optic neuropathy is caused by theinflammation of vessels supplying blood to the optic nerves, known astemporal arteritis. This condition usually presents with sudden andsevere vision loss in one eye, pain in the jaw with chewing, tendernessin the temple area, loss of appetite, and a generalized felling offatigue or illness.

“Macular degeneration” refers to the physical disturbance of the centerof the retina called the macula, leading to a loss of central vision,although color vision and peripheral vision may remain clear. Visionloss usually occurs gradually and typically affects both eyes atdifferent rates.

A “demyelinating disorder” is a condition resulting from damage to themyelin sheath, which surrounds nerves and is responsible for efficienttransmission of nerve impulses to the brain. A demyelinating disordermay result in muscle weakness, poor coordination and possible paralysis.Examples of demyelinating disorders include, but are not limited to:multiple sclerosis, optic neuritis, transverse neuritis andGuillain-Barré syndrome. When treating a demyelinating disorder, acomposition according to the present invention may include anN-methyl-D-aspartate-type glutamate receptor (NMDAR) antagonist (e.g.,memantine) or beta interferon isoforms, copaxone or Antegren(natalizumab). Since neuronal damage may occur in demyelantingconditions such as multiple sclerosis, useful drug compositions may alsoprotect the neuron instead of or in addition to the myelin.

“Multiple sclerosis” refers to a chronic disease of the central nervoussystem, which predominantly affects young adults and is characterized byareas of demyelination and T-cell predominant perivascular inflammationin the white matter of the brain. Some axons may be spared from thesepathological processes. The disease begins most commonly with acute orsubacute onset of neurologic abnormalities. Initial and subsequentsymptoms may dramatically vary in their expression and severity over thecourse of the disease, which usually lasts for many years. Earlysymptoms may include numbness and/or paresthesia, mono- or paraparesis,double vision, optic neuritis, ataxia and bladder control problems.Subsequent symptoms also include more prominent upper motor neuronsigns, i.e., increased spasticity, increasing para- or quadriparesis.Vertigo, incoordination and other cerebellar problems, depression,emotional lability, abnormalities in gait, dysarthria, fatigue and painare also commonly seen.

“Sequelae of hyperhomocystinemia” refers to a condition following as aconsequence hyperhomocystinemia, i.e., elevated levels of homocysteine.

“Convulsion” refers to a violent involuntary contraction or series ofcontractions of the muscles.

“Pain” refers to an unpleasant sensation associated with actual orpotential tissue damage that is mediated by specific nerve fibers to thebrain where its conscious appreciation may be modified by variousfactors. See, e.g., Mosby's Medical, Nursing & Allied Health Dictionary,5^(th) edition (1998); and Stedman's Medical Dictionary, 25^(th) edition(1990).

“Anxiety” refers to a state of apprehension, uncertainty, and/or fearresulting from the anticipation of a realistic or fantasized threateningevent or situation, often impairing physical and psychologicalfunctioning.

“Schizophrenia” refers to any of a group of psychotic disorders usuallycharacterized by withdrawal from reality, illogical patterns ofthinking, delusions, and hallucinations, and accompanied in varyingdegrees by other emotional, behavioral, or intellectual disturbances.Schizophrenia is associated with dopamine imbalances in the brain anddefects of the frontal lobe.

“Muscle spasm” refers to an often painful involuntary muscularcontraction.

“Migraine headache” refers to a severe, debilitating headache oftenassociated with photophobia and blurred vision.

“Urinary incontinence” refers to the inability to control the flow ofurine and involuntary urination.

“Nicotine withdrawal” refers to the withdrawal from nicotine, anaddictive compound found in tobacco, which is characterized by symptomsthat include headache, anxiety, nausea and a craving for more tobacco.Nicotine creates a chemical dependency, so that the body develops a needfor a certain level of nicotine at all times. Unless that level ismaintained, the body will begin to go through withdrawal.

“Opiate tolerance” refers to a homeostatic response that reduces thesensitivity of the system to compensate for continued exposure to highlevels of an opiate, e.g., heroine or morphine. When the drug isstopped, the system is no longer as sensitive to the soothing effects ofthe enkephalin neurons and the pain of withdrawal is produced.

“Opiate withdrawal” refers to an acute state caused by cessation ordramatic reduction of use of opiate drugs that has been heavy andprolonged (several weeks or longer). Opiates include heroin, morphine,codeine, Oxycontin, Dilaudid, methadone and others. Opiate withdrawaloften includes sweating, shaking, headache, drug craving, nausea,vomiting, abdominal cramping, diarrhea, inability to sleep, confusion,agitation, depression, anxiety, and other behavioral changes.

“Emesis” refers to the act of vomiting.

“Brain edema” refers to an excessive accumulation of fluid in, on,around and/or in relation to the brain.

“AIDS- (or HIV-)induced (or associated) dementia” refers to dementia (adeterioration of intellectual faculties, such as memory, concentration,and judgment, resulting from an organic disease or disorder of thebrain) induced by human immunodeficiency virus (HIV), which causesacquired immunodeficiency syndrome (AIDS).

“HIV-related neuropathy” refers to a neuropathy in a mammal infectedwith HIV where the neuropathy is caused by infections such as with CMVor other viruses of the herpes family. Neuropathy is the name given to agroup of disorders whose symptoms may range from a tingling sensation ornumbness in the toes and fingers to pain to paralysis.

“Ocular damage” refers to any damage to the eyes or in relation to theeyes.

“Retinopathy” refers to any pathological disorder of the retina.

“Cognitive disorder” refers to any cognitive dysfunction, for example,disturbance of memory (e.g., amnesia) or learning.

In another embodiment of the invention, neuroprotective compounds of thepresent invention are also used to treat aging due to freeradical-induced damage, for example, to slow the process of normal agingof the nervous system of an organism and its symptoms (Finkel, Nat. Rev.Mol. Cell Biol. 6:971-976, 2005; Finkel et al., Nature 408:239-247,2000). Accordingly, compositions are provided that comprise an amount ofa compound according to the present invention that is effective to slowthe process of aging of the nervous symptom or a symptom thereof in anindividual.

Neurotoxic and Neuroprotective Electrophiles

Reaction of some electrophiles (referred to herein as “neurotoxic”electrophiles) with reduced cysteine residues, e.g., those ofglutathione (GSH), can induce neurotoxicity by decreasing the reductivecapacity of the cell (Suzuki. et al., J. Am. Chem. Soc. 119:2376-2385,1997; Spencer et al., FEBS Lett. 24:246-250, 1994). Thus, earlierstudies focused on the neurotoxic effects of endogenous electrophilessuch as 15d-PGJ₂ (Shibata et al., J. Biol. Chem. 281:1196-1204, 2005),catecholamine metabolites (including dopamine) (Spencer et al., FEBSLett. 24:246-250, 1994), and anti-tumor agents (including doxorubicin)(Wetzel et al., Eur. J. Neurosci. 18:1050-1060, 2003). In this regard,electrophiles can contribute to neuronal death by several mechanisms:(1) GSH depletion, (2) reactive oxygen species (ROS) production, (3) DNAdamage, (4) p53 activation, (5) Fas/Fas ligand induction, and (6)mitochondrial dysfunction (Suzuki. et al., J. Am. Chem. Soc.119:2376-2385, 1997; Spencer et al., FEBS Lett. 24:246-250, 1994;Shibata et al., J. Biol. Chem. 281:1196-1204, 2005; Wetzel et al., Eur.J. Neurosci. 18:1050-1060, 2003). Alkylation of GSH cysteines byelectrophiles (Suzuki. et al., J. Am. Chem. Soc. 119:2376-2385, 1997)depletes the reducing capability of the cell (Suzuki. et al., J. Am.Chem. Soc. 119:2376-2385, 1997; Shibata et al., J. Biol. Chem.281:1196-1204, 2005; Wetzel et al., Eur. J. Neurosci. 18:1050-1060,2003), and simultaneously the alkylated complex is extruded through thecell membrane via the multidrug resistance-associated protein-1 (MRP-1)(Sekine et al., Am. J. Physiol. Renal Physiol. 290:F251-F261, 2006).Accumulation of reactive oxygen species (ROS) precipitated by GSHdepletion contributes to mitochondrial dysfunction, which activatesapoptotic machinery, resulting in cytochrome c release, Baxtranslocation to the inner mitochondrial membrane, and caspaseactivation (Shibata et al., J. Biol. Chem. 281:1196-1204, 2005; Wetzelet al., Eur. J. Neurosci. 18:1050-1060, 2003). In addition, alkylationof guanine residues inhibits the transcription and replication of DNA,and activates the p53-dependent apoptotic pathway (Spencer et al., FEBSLett. 24:246-250, 1994; Shibata et al., J. Biol. Chem. 281:1196-1204,2005).

On the other hand, in response to electrophiles some cells mount an“electrophile counterattack,” a system that detoxifies electrophiles andremoves them immediately (Eggler et al., Proc. Natl. Acad. Sci. USA102:10070-10075, 2005; Dinkova-Kostova et al., Chem. Res. Toxicol.18:1779-1791, 2005; Talalay, Biofactors 12:5-11, 2000; Hong et al.,Chem. Res. Toxicol. 18:1917-1926, 2005; Padmanabhan et al., Mol. Cell21:689-700, 2006). The electrophilic counterattack usually liesrelatively dormant but becomes activated by electrophiles themselves(Dinkova-Kostova et al., Chem. Res. Toxicol. 18:1779-1791, 2005;Talalay, Biofactors 12:5-11, 2000; Hong et al., Chem. Res. Toxicol.18:1917-1926, 2005; Padmanabhan et al., Mol. Cell 21:689-700, 2006).Since this electrophile counterattack eliminates not only theelectrophiles but also ROS, it can thus prevent neurodegeneration andtumor growth (Dinkova-Kostova et al., Chem. Res. Toxicol. 18:1779-1791,2005; Talalay, Biofactors 12:5-11, 2000; Hong et al., Chem. Res.Toxicol. 18:1917-1926, 2005; Padmanabhan et al., Mol. Cell 21:689-700,2006). Thus, electrophiles could possibly be used as both anti-tumor(Dinkova-Kostova et al., Chem. Res. Toxicol. 18:1779-1791, 2005;Talalay, Biofactors 12:5-11, 2000; Hong et al., Chem. Res. Toxicol.18:1917-1926, 2005; Padmanabhan et al., Mol. Cell 21:689-700, 2006) andneuroprotective agents (Satoh et al., Proc. Natl. Acad. Sci. USA103:768-773, 2006; Shih et al., J. Neurosci. 25:10321-10335, 2005; Kraftet al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.23:3394-3406, 2003). Talalay (Biofactors 12:5-11, 2000) was the first tointroduce this concept and termed the phenomenon “chemoprevention” inview of its cancer-combating properties. Many chemopreventive agents areelectrophilic and increase cellular resistance to oxidative stress(Dinkova-Kostova et al., Chem. Res. Toxicol. 18:1779-1791, 2005;Talalay, Biofactors 12:5-11, 2000; Hong et al., Chem. Res. Toxicol.18:1917-1926, 2005; Padmanabhan et al., Mol. Cell 21:689-700, 2006).This form of chemoprevention often entails a transcription-basedmechanism involving a specific signaling pathway (the Keap1/Nrf2pathway) and the induction of phase 2 genes, which encode enzymesrepresenting a coordinated response to electrophiles, including thegenes encoding the following enzymes: heme oxygenase 1 (HO-1), whichgenerates antioxidants (bilirubin); NADPH-quinone oxidoreductase (NQO1),which reduces quinones to hydroquinones; multidrug resistance-associatedprotein (MRP-1), which transports GSH-conjugated compounds out of thecell; γ-glutamylcysteine synthetase (γ-GCS), which is involved insynthesis of GSH; and cysteine/glutamate antiporter (xCT), which isinvolved in uptake of cystine, a precursor of cysteine. Other phase 2enzymes include: glutathione S-transferase, which conjugateselectrophiles to GSH; catalase, which detoxifies hydrogen peroxide;manganese superoxide dismutase, which detoxifies superoxide; andmetallothionein-1 and -2, which detoxify heavy metals. Most phase 2enzymes are involved in drug detoxification and redox regulation and areinduced by electrophilic compounds (Dinkova-Kostova et al., Chem. Res.Toxicol. 18:1779-1791, 2005; Talalay, Biofactors 12:5-11, 2000; Hong etal., Chem. Res. Toxicol. 18:1917-1926, 2005; Padmanabhan et al., Mol.Cell 21:689-700, 2006).

In neurons, electrophiles also manifest two disparate actions: aneurotoxic effect, mediated by a decrease in total cellular reductivecapacity, but also an electrophile counterattack via the induction ofphase 2 genes. If the neurotoxic effects predominate, then a particularelectrophile will kill a neuron [e.g., doxorubicin (Wetzel et al., Eur.J. Neurosci. 18:1050-1060, 2003] and menadione [Nguyen et al., Antioxid.Redox Signal. 5:629-634, 2003)]. In contrast, if the electrophilecounterattack predominates, which occurs especially in response to weakelectrophiles, then the electrophilic response will rescue neurons fromfree radical-related insults [e.g., as observed with tert-butylhydroquinone (TBHQ) (Shih et al., J. Neurosci. 25:10321-10335, 2005;Kraft et al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.23:3394-3406, 2003) and neurite outgrowth-promoting prostaglandin (NEPP)compounds. Thus, preferential activation of the electrophilecounterattack, while minimizing the neurotoxic effects of electrophilesthat deplete total cellular redox state, has been touted as a newtherapeutic strategy against neurodegeneration (Satoh et al., Proc.Natl. Acad. Sci. USA 103:768-773, 2006; Shih et al., J. Neurosci.25:10321-10335, 2005; Kraft et al., J. Neurosci. 24:1101-1112, 2004;Shih et al., J. Neurosci. 23:3394-3406, 2003). Murphy et al. (J.Neurochem. 56:990-995, 1991) first demonstrated that exogenouselectrophiles can be neuroprotective. For example, TBHQ and dimethylfumarate protected neurons against oxidative stress. This protectiveeffect was associated with the induction of NADPH-quinoneoxidoreductase-1 (NQO1), a phase 2 enzyme (Murphy et al., J. Neurochem.56:990-995, 1991; Shih et al., J. Biol. Chem. 280:22925-22936, 2005).Electrophilic neuroprotection displays the following parameters: (1) theprotection is transcription dependent and thus requires pretreatment;(2) the protective compounds themselves may be electrophiles or maygenerate electrophiles; (3) the compounds spare essential cellular redoxfactors such as GSH; and (4) the compounds induce expression of phase 2enzymes, such as NQO1 and heme oxygenase-1 (HO-1), often via theKeap1/Nrf2 transcription factor pathway (Satoh et al., Proc. Natl. Acad.Sci. USA 103:768-773, 2006; Shih et al., J. Neurosci. 25:10321-10335,2005; Kraft et al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J.Neurosci. 23:3394-3406, 2003). Interestingly, Murphy, Johnson andcolleagues showed that the neuroprotective effect of TBHQ against strokeis mediated via activation of the Keap1/Nrf2 pathway in astrocytes and aresulting paracrine effect on neurons when it is used at properconcentrations (Shih et al., J. Neurosci. 25:10321-10335, 2005; Kraft etal., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.23:3394-3406, 2003; Shih et al., J. Biol. Chem. 280:22925-22936, 2005).In contrast, doxorubicin and menadione are neurotoxic quinine-basedelectrophilic compounds; they deplete intracellular GSH and kill neuronsby precipitating oxidative stress at virtually any concentration (Wetzelet al., Eur. J. Neurosci. 18:1050-1060, 2003; Nguyen et al., Antioxid.Redox Signal. 5:629-634, 2003). Thus, electrophiles can be divided intotwo groups: neurotoxic electrophiles and neuroprotective electrophiles.At low concentration the NEPP family of electrophiles, particularlyNEPP11, protects neurons against oxidative stress (Example 1; see alsoSatoh et al., J. Neurochem. 77:50-62, 2001). NEPP compounds that weregenerated on the basis of the chemical structure of cyclopentenoneprostaglandins protect neurons from oxidative stress in anHO-1-dependent manner (Satoh et al., J. Neurochem. 77:50-62, 2001; Satohet al., J. Neurochem. 75:1092-1102, 2000; Satoh et al., Eur. J.Neurosci. 17:2249-2255, 2003). We reasoned that since NEPPs wereelectrophilic compounds, the key to an understanding of theirneuroprotective action was identification of the protein thiols withwhich they reacted. Along these lines, it was found that a number ofelectrophiles exerted neuroprotective effects that were closely linkedwith the Keap1/Nrf2 transcriptional pathway (Example 1; see also Shih etal., J. Neurosci. 25:10321-10335, 2005; Kraft et al., J. Neurosci.24:1101-1112, 2004; Shih et al., J. Neurosci. 23:3394-3406, 2003). Asecond important point, however, is that this class of molecules ispreferentially concentrated in neurons and thus works directly onneurons in a targeted fashion.

Concerning the chemical reactions and signal transduction pathwaystriggered by NEPPs, electrophile binding to cysteine residue(s) caninitiate neuroprotection, based on the following lines of evidence: (1)NEPP and related compounds bind to cysteine in a cell-free system(Suzuki. et al., J. Am. Chem. Soc. 119:2376-2385, 1997); (2)N-ethylmaleimide, a sulfhydryl alkylating agent, abolishes the bindingof NEPP compounds to the cysteine residues of bovine serum albumin(Example 1); (3) cross-conjugated dienone, the electrophilic moiety ofNEPPs, is required for neuroprotection and HO-1 induction (Satoh et al.,J. Neurochem. 77:50-62, 2001; Satoh et al., J. Neurochem. 75:1092-1102,2000); (4) Keap1 mutants (cysteine at position 151 replaced by serine)abolishe HO-1 induction by NEPP11 and its neuroprotective effect(Example 1). Thus, alkylation/redox signaling of Keap1 by NEPP11 mightinitiate neuroprotective events through induction of phase 2 genes(Example 1; Satoh et al., Eur. J. Neurosci. 17:2249-2255, 2003).

A number of studies have provided evidence that electrophiles canprotect neurons via activation of the Keap1/Nrf2 pathway with theconsequent induction of HO-1 and other phase 2 enzymes. These enzymesengender neuroprotection via modulation of the intracellular redox state(Example 1; Shih et al., J. Neurosci. 25:10321-10335, 2005; Kraft etal., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.23:3394-3406, 2003). The antioxidant response element (ARE) is atranscriptional element that is located in the 5′ upstream promoterregion of genes that encode phase 2 enzymes. Transcription factorsbinding to AREs thus mediate the induction of phase 2 enzymes(Dinkova-Kostova et al., Chem. Res. Toxicol. 18:1779-1791, 2005;Talalay, Biofactors 12:5-11, 2000; Hong et al., Chem. Res. Toxicol.18:1917-1926, 2005; Padmanabhan et al., Mol. Cell 21:689-700, 2006).Yamamoto's group were the first to demonstrate that the Keap1/Nrf2pathway activates AREs (Padmanabhan et al., Mol. Cell 21:689-700, 2006).Keap1 is an adapter protein for ubiquitination of Nrf2 and thus drivesthe continuous degradation of this transcription factor (Eggler et al.,Proc. Natl. Acad. Sci. USA 102:10070-10075, 2005; Dinkova-Kostova etal., Chem. Res. Toxicol. 18:1779-1791, 2005; Talalay, Biofactors12:5-11, 2000; Hong et al., Chem. Res. Toxicol. 18:1917-1926, 2005;Padmanabhan et al., Mol. Cell 21:689-700, 2006). When electrophilesreact with critical cysteine residues on Keap1 to form an adduct, theyperturb this system and stabilize Nrf2, causing the liberation of Nrf2and allowing it to be translocated into the nucleus, where it binds toAREs and stimulates the expression of phase 2 genes (Eggler et al.,Proc. Natl. Acad. Sci. USA 102:10070-10075, 2005; Dinkova-Kostova etal., Chem. Res. Toxicol. 18:1779-1791, 2005; Talalay, Biofactors12:5-11, 2000; Hong et al., Chem. Res. Toxicol. 18:1917-1926, 2005;Padmanabhan et al., Mol. Cell 21:689-700, 2006). In fact, Nrf2 is aprimary transcription factor responsible for the electrophilecounterattack response in the brain (Satoh et al., Proc. Natl. Acad.Sci. USA 103:768-773, 2006; Shih et al., J. Neurosci. 25:10321-10335,2005; Kraft et al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J.Neurosci. 23:3394-3406, 2003; Johnson et al., J. Neurochem.81:1233-1241, 2002, Lee et al., J. Biol. Chem. 278:37948-37956, 2003).Experiments using ARE reporter mice have demonstrated that TBHQactivates AREs in astroglial cells, resulting in activation of theKeap1/Nrf2 pathway and subsequent protection of neurons from oxidativeinsult; thus, TBHQ protects neurons by activating the Keap1/Nrf2 pathwayin astrocytes (Shih et al., J. Neurosci. 25:10321-10335, 2005; Kraft etal., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.23:3394-3406, 2003; Johnson et al., J. Neurochem. 81:1233-1241, 2002;Lee et al., J. Biol. Chem. 278:37948-37956, 2003). Experiments usingcerebral cortical cultures from Nrf2 knockout mice confirmed theimportance of Nrf2 protein for induction of phase 2 genes by TBHQ inastrocytes and the consequent neuroprotective effects (Johnson et al.,J. Neurochem. 81:1233-1241, 2002, Lee et al., J. Biol. Chem.278:37948-37956, 2003).

Our research suggests a pathway for electrophile-inducedneuroprotection. An electrophile binds to the cytosolic regulatorprotein Keap1, which in turn liberates the transcription factor Nrf2.Nrf2 then translocates into the nucleus, where it activates ARE sites onthe HO-1 promoter. Transcription of HO-1 is thus activated, and theresulting increase in HO-1 protein leads to degradation of hememolecules, producing biliverdin and ultimately bilirubin. Theaccumulation of bilirubin, a potent antioxidant molecule, mediates atleast in part the neuroprotective effect of HO-1. After exposure to theelectrophile THBQ, this pathway is triggered in astrocytes, but afterexposure to NEPP compounds, the pathway is activated in neurons.

Electrophilic NEPP compounds accumulate in neurons rather thanastrocytes to activate the Keap1/Nrf2 pathway and induce phase 2 genessuch as HO-1 directly in neurons (Satoh et al., Proc. Natl. Acad. Sci.USA 103:768-773, 2006). The apparent difference in the cell typemanifesting activated Keap1/Nrf2 after TBHQ versus NEPP exposure maypossibly be attributed to the disparate chemical structures of thesecompounds, which can differentially affect their cellular uptake (Satohet al., Proc. Natl. Acad. Sci. USA 103:768-773, 2006). (Catechol-typeelectrophilic compounds, such as those listed in Table 1, typicallyenter into both neurons and astrocytes.)

Since plants produce a vast variety of electrophiles, some foods maycontain chemopreventive compounds (Talalay, Biofactors 12:5-11, 2000).For example, curcumin, the powdered rhizome of Curcuma longa Linn,represents one of many potential electrophiles of plant origin that actas neuroprotective agents (Talalay, Biofactors 12:5-11, 2000). Curcuminactivates the Keap1/Nrf2/HO-1 pathway and exerts protective effectsagainst brain ischemia (Balogun et al., Biochem. J. 371:887-895, 2003).Curcumin also protects neurons against chronic neurodegeneration in amurine model of Alzheimer's disease (Lim et al., J. Neurosci.21:8370-8377, 2001). Additionally, curcumin displays anti-tumor(pro-apoptotic) effects (Talalay, Biofactors 12:5-11, 2000). Based onthis background information, Dinkova-Kostova et al. (Proc. Natl. Acad.Sci. USA 98:3404-3409, 2001) screened analogues of curcumin forinduction of NQO1 activity and found thatbis(4-hydroxybenzylidene)acetone (4-HBA) had the most potentanti-carcinogenic activity (Dinkova-Kostova et al., Proc. Natl. Acad.Sci. USA 98:3404-3409, 2001). Interestingly, our group (Satoh et al., J.Neurochem. 77:50-62, 2001) independently reached a similar conclusionregarding this class of chemical structures, i.e., cross-conjugateddienones, because of the activity of NEPP11 in promoting neuronalsurvival. Thus, one structural class of neuroprotective electrophilescontains a cross-conjugated dienone.

One phase 2 gene product induced by electrophilic activation of theKeap1/Nrf2 pathway is HO-1. This enzyme oxidatively cleaves heme tobiliverdin, forming carbon monoxide (CO) and releasing chelated Fe²⁺(Maines and Gibbs, Biochem. Biophys. Res. Commun. 338:568-577, 2005).Bilirubin, the product of reduction of biliverdin, serves as a potentfree radical scavenger (Stocker, Antioxid. Redox Signal. 6:841-849,2004). HO-1 plays an obligatory role in resistance to oxidative stressas revealed by reports that fibroblasts from HO-1^(−/−) mice aresensitive to oxidative stress (Poss and Tonegawa, Proc. Natl. Acad. Sci.USA 94:10925-10930, 1997) while cerebellar granule neurons from HO-1transgenic mice are resistant to oxidative stress (Chen et al., J.Neurochem. 75:304-313, 2000). Among phase 2 enzymes, HO-1 has attractedspecial attention because of its therapeutic effects, for instance,against inflammation (Lee et al., Nat. Med. 8:240-246, 2002). Barananoand Snyder (Proc. Natl. Acad. Sci. USA 98:10996-1002, 2001), Maines andGibbs (Biochem. Biophys. Res. Commun. 338:568-577, 2005), and our group(Example 1) have all proposed that HO-1 inducers are neuroprotective.For example, as demonstrated in Example 1, the HO-1 protein plays acentral role in the neuroprotective effect of NEPP11, as evident fromfollowing facts: (1) HO-1 is dramatically increased by NEPP11 (Example1; Satoh et al., J. Neurochem. 75:1092-1102, 2000); (2) HO-1 inhibitorsabrogate the neuroprotective effect of NEPP11 (Example 1); (3)transfection with HO-1 cDNA is neuroprotective (Satoh et al., J.Neurochem. 75:1092-1102, 2000); and bilirubin, which is downstream fromHO-1 enzymatic activity, is also neuroprotective (Satoh et al., J.Neurochem. 75:1092-1102, 2000).

Furthermore, as stated above, NEPP compounds accumulate in neurons, andHO-1 is induced in cortical neurons after intraperitoneal injection ofmice with NEPP11. Thus, induction of HO-1 by treatment with NEPP11 orsimilar agents represents a novel method of targeted neuronal therapyfor neurodegenerative disorders (Example 1). HO exists as 2 isozymes:HO-1, an inducible form, and HO-2, a constitutive form (Maines andGibbs, Biochem. Biophys. Res. Commun. 338:568-577, 2005). HO-2 activityis also essential for protecting neurons against oxidative stress, asevidenced by studies on HO-2 knockout mice (Dore. et al., Proc. Natl.Acad. Sci. USA 96:2445-3450, 1999). We have proposed that HO-1 and HO-2play central roles in protecting neurons against oxidative stress butvia differential regulation: HO-2 is activated first by phosphorylation,and HO-1 is activated subsequently via transcriptional mechanisms (Satohet al., Eur. J. Neurosci. 17:2249-2255, 2003).

Neuroprotective electrophiles often increase basal levels of GSH (Sun etal., Biochem. Biophys. Res. Commun. 14:371-377, 2005). Since GSH is amajor reducing substance protecting against cellular oxidative stress,an increase in its level could in part account for the neuroprotectiveeffects of electrophiles (Sun et al., Biochem. Biophys. Res. Commun.14:371-377, 2005). The increase in GSH is due to an increase in bothcystine (a precursor of cysteine) uptake and GSH synthesis. The AREregulates expression of both the cystine/glutamate antiporter (xCT) andγ-glutamylcysteine synthetase (γ-GCS), which represent the rate-limitingsteps for cystine uptake and GSH synthesis, respectively (Talalay,Biofactors 12:5-11, 2000). Thus, electrophilic induction of xCT andγ-GCS via the ARE may contribute to neuroprotection by increasing GSH.

Additionally, NQO1 is a phase 2 gene product induced by electrophiles.NQO1 catalyzes the two-electron reduction of several quinones to thecorresponding hydroquinone (Talalay, Biofactors 12:5-11, 2000;Dinkova-Kostova et al., Proc. Natl. Acad. Sci. USA 98:3404-3409, 2001).Although reduced quinones could potentially function as effectiveantioxidants, other evidence suggests that it is unlikely that NQO1activity is involved in the neuroprotective effect of electrophiles forthe following reasons: (1) transfection of neuronal cells with the NQO1gene does not offer protection (Murphy et al., J. Neurochem. 56:990-995,1991), and (2) NQO1 may paradoxically enhance neuronal cell death by anunknown mechanism (Kapinya et al., J. Neurochem. 84:1028-1039, 2003).

TBHQ and NEPP11 preferentially activate the electrophilic counterattackwhile minimizing neurotoxic effects due to the binding of TBHQ andNEPP11 to cysteines specific for neuroprotection. One such cysteineresidue appears to be Cys 151 of Keap1 (Eggler et al., Proc. Natl. Acad.Sci. USA 102:10070-10075, 2005; Hong et al., Chem. Res. Toxicol.18:1917-1926, 2005); such cysteine thiols are potential drug targets forthe development of novel neuroprotective agents againstneurodegenerative diseases.

It is possible that, when systemically administered, electrophiles suchas NEPP11 and 4-HBA and thiols may react before the electrophiles reachtheir intended targets in the brain. Thus, a compound that acts as apro-drug and converts to an electrophile by oxidation upon reaching theintended target may be desirable. For example, terpenoids that possesscatechol rings represent pro-electrophilic compounds that can beconverted to quinone-type electrophiles by oxidation (Dinkova-Kostova etal., Proc. Natl. Acad. Sci. USA 102:4584-4589, 2005). In Parkinson'sdisease, oxidative stress plays a critical role in disease progression(Jener, Ann. Neurol. 53:S36-S38, 2003) but could be used to activatepro-electrophilic compounds via their oxidation at the target site toprovide neuroprotection where it is needed. This approach represents anovel strategy against neurodegenerative disorders that could activateelectrophilic drugs via pathological activity.

Carnosic acid (CA) is a polyphenolic antioxidant derived from the plantsrosemary (Rosmarinus officinalis) and sage (Salvia officinalis L.), forexample. We have found that carnosic acid (CA) is a terpenoid that canbe converted to a neuroprotective quinone by oxidation. It activates theKeap1/Nrf2 pathway similar in a manner similar to NEPPs. CA has certainadvantages as well. CA is brain permeable. It is also activated byoxidation and hence may be activated, i.e., converted to itsneuroprotective quinone metabolite, at the site of the insult.Furthermore, it will remain for longer periods in the injured tissue. Invitro, approximately 0.1 μM to 10 μM CA is neuroprotective. In vivo, adosage range of between 1 mg/kg to 100 mg/kg is expected to be optimal,although other dosages may be used as well. The precise dosage that isuseful in the practice of the present invention may be determinedwithout undue experimentation. Methods for obtaining carnosic acid fromrosemary and sage are taught, for example, in U.S. Pat. Nos. 5,256,700;5,859,293; and 6,335,373, and the purified compound is availablecommercially.

Carnosic acid derivatives may also be used in the practice of thepresent invention. See, for example, U.S. Pat. No. 6,479,549 for someexamples of carnosic acid derivatives and their chemical synthesis.Other CA derivatives are known in the art or may be produced by theskilled artisan without undue experimentation. Examples include thefollowing compounds and derivatives thereof in which the benzene ringhas two hydroxyl groups in para orientation rather than orthoconfiguration (by CA Index Name): 1,4a(2H)-Phenanthrenedicarboxylicacid,1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1-methyl-7-(1-methylethyl)-,(1R,4aR,10aS)-; 1,4a(2H)-Phenanthrenedicarboxylic acid,1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1-methyl-7-(1-methylethyl)-,1-methyl ester, (1R,4aR,10aS)-; 4a(2H)-Phenanthrenecarboxylic acid,1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1-(hydroxymethyl)-1-methyl-7-(1-methylethyl)-,(1R,4aR,10aS)-; 4a(2H)-Phenanthrenecarboxylic acid,1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-7-(2-hydroxy-1-methylethyl)-1,1-dimethyl-,[4aR-[4aα,7(R*),10aβ]]-(9CI) (also called 16-hydroxycarnosic acid);5ξ,10ξ-Podocarpa-8,11,13-trien-17-oic acid,7,11,12-trihydroxy-13-isopropyl (7CI); 4a(2H)-Phenanthrenecarboxylicacid,1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1,1-dimethyl-7-(1-methylethyl)-9-oxo-,(4aR-trans)-(9CI); 4a(2H)-Phenanthrenecarboxylic acid,1,3,4,9,10,10a-hexahydro-5,6,9-trihydroxy-1,1-dimethyl-7-(1-methylethyl)-10-oxo-,[4aR-(4aα,9β,10aβ)]-(9CI); 4a(2H)-Phenanthrenecarboxylic acid,1,3,4,9,10,10a-hexahydro-5,6,9-trihydroxy-1,1-dimethyl-7-(1-methylethyl)-,(4aR,9S,10aS)- (also called Carnosolic acid); and4a(2H)-Phenanthrenecarboxylic acid,7-[2-(acetyloxy)-1-methylethyl]-1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1,1-dimethyl-(9CI).

Screening Methods

A number of NEPPs and other electrophilic and pro-electrophiliccompounds are known in the literature.

Methods are provided for screening for other substances that areeffective for neuroprotection, as described in detail in the Examples.In addition to the methods taught herein, methods for screening forneuroprotective substances are known in the art.

Pharmaceutical Compositions and Methods.

The compositions of the invention can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient, in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Such compositions may be systemically administered in vivo by a varietyof routes. For example, they may be administered orally, in combinationwith a pharmaceutically acceptable excipients such as an inert diluentor an assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraladministration, the active ingredient or ingredients may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of active ingredient in such useful compositions is such that aneffective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The compositions may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of aneuroprotective compound according to the present invention, their saltsor solvates, and other active ingredients can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating aneuroprotective compound according to the present invention or otheractive ingredients in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, a neuroprotective compound according to thepresent invention and other active ingredients may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of a neuroprotective compound according to the presentinvention or other active ingredients can be determined by comparingtheir in vitro activity and in vivo activity in animal models. Methodsfor the extrapolation of effective dosages in mice, and other animals,to humans are known to the art; for example, see U.S. Pat. No.4,938,949.

Generally, the concentration of a neuroprotective compound according tothe present invention or other active ingredients of the invention in aliquid composition will be from about 0.1-25 wt-%, preferably from about0.5-10 wt-%. The concentration in a semi-solid or solid composition suchas a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use alone or with other agents will vary not only with theparticular salt selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician.

In general, however, a suitable dose may be in the range of from about0.5 to about 100 mg/kg, e.g., from about 1 to about 75 mg/kg of bodyweight per day, or 1.5 to about 50 mg per kilogram body weight of therecipient per day, or about 2 to about 30 mg/kg/day, or about 2.5 toabout 15 mg/kg/day.

The compound may be conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

The active ingredient may be administered to achieve peak plasmaconcentrations of the active compound of from about 0.5 to about 75 μM,preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM.This may be achieved, for example, by the intravenous injection of a0.05 to 5% solution of the active ingredient, optionally in saline, ororally administered as a bolus containing about 1-100 mg of the activeingredient. Desirable blood levels may be maintained by continuousinfusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Pharmaceutical compositions according to the invention may comprise oneor more than one neuroprotective substance according to the invention.Pharmaceutical compositions comprising a neuroprotective compoundaccording to the present invention may also include other activeingredients.

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

The present invention will be further described by the followingnonlimiting examples.

Example 1

A recent study showed that activation of the Keap1/Nrf2/ARE pathwaymediates HO-1 induction by electrophiles (Itoh et al., Mol. Cell. Biol.24:36-45, 2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002).Thus we focused on this pathway as a possible mechanism by which neuriteoutgrowth-promoting prostaglandins (NEPPs) promote HO-1 induction andneuroprotection. We have found that NEPPs protect cortical neurons bothin vitro and in vivo against neuronal degeneration by acting aselectrophiles to activate the Keap1/Nrf2 pathway.

Materials and Methods

Cell Cultures, Transfection, and Glutathione (GSH) Measurement.

HT22 cells (Satoh et al., Eur. J. Neurosci. 17:2249-2255, 2003; Satoh etal., J. Neurochem. 77:50-62, 2001; Sagara et al., J. Biol. Chem.277:36204-36215, 2002) and primary cortical neurons (Bonfoco et al.,Proc. Natl. Acad. Sci. USA 92:7162-7166, 1995) were cultured asdescribed. Transfection was performed with Lipofectamine 2000(Invitrogen). In the reporter gene assays, firefly luciferase activityin cell lysates was measured with a luminometer (Promega). Total GSH(reduced and oxidized) was determined as described (Lee et al., Biochem.Biophys. Res. Commun. 280:286-292, 2001). Immunoprecipitation, westernblots, and immunofluorescence. These assays were performed as described(Gu et al., Science 297:1186-1190, 2002) by using the followingantibodies: anti-HO-1 (SPA895, 1:1000, Santa Cruz Biotechnology),anti-Keap1 (1:100, Santa Cruz Biotechnology), or anti-actin (1:5,000,Oncogene Research Products, San Diego).

Electrophoretic Mobility Shift Assays (EMSAs).

Double-stranded antioxidant-responsive elements (AREs) were labeled byusing a biotin 3′-end DNA labeling kit (Pierce). Nuclear lysates wereincubated with the labeled probe for 20 min at room temperature,resolved on an 8% native polyacrylamide gel, and transferred toHybond-N⁺ (Amersham Pharmacia). Signals were visualized withperoxidase-conjugated streptavidin (Pierce).

Focal Cerebral Ischemia and Reperfusion.

The filament model of middle cerebral artery occlusion(MCAO)/reperfusion was used as described (Gu et al., Science297:1186-1190, 2002; see Supporting Methods in Supporting Text).

Statistical Analysis.

Experiments presented were repeated at least three times with foursamples. The data are presented as mean±standard deviation (SD) (for invitro experiments) or SEM (for in vivo experiments).

Results

Thiols as Targets of Electrophilic NEPPs.

We generated NEPP-related compounds based on the chemical structures ofcyclopentenone prostaglandins and found that NEPP6 and -11 protectedneurons against oxidative stress (Satoh et al., Eur. J. Neurosci.17:2249-2255, 2003; Satoh et al., J. Neurochem. 77:50-62, 2001) and thatinduction of HO-1 played an essential role in these neuroprotectiveeffects (Satoh et al., Eur. J. Neurosci. 17:2249-2255, 2003). NEPP11afforded more potent neuroprotection than did NEPP6, probably becauseNEPP11 is more lipophilic, allowing for better CNS permeability (Satohet al., J. Neurochem. 77:50-62, 2001). The cross-conjugated dienonestructure of NEPPs is critical for their biological effects (Satoh etal., J. Neurochem. 77:50-62, 2001) and underlies the electrophilicity ofcarbon #11 and thus its high chemical reactivity with thiols (Satoh etal., J. Neurochem. 77:50-62, 2001).

Through its single free thiol group (on cysteine residue #34), BSA hasbeen used as an in vitro example for adduct formation by electrophiliccompounds. We tested whether the free cysteine of bovine serum albumin(BSA) could form an adduct with NEPP compounds. If carbon #11 on theNEPP compounds binds to this free cysteine of BSA, then pretreatmentwith N-ethylmaleimide (NEM), in irreversible thiol alkylating agent,would abolish this binding. In an experiment to test this idea, wesynthesized NEPP6-biotin (Satoh et al., J. Neurochem. 77:50-62, 2001).Biotin was conjugated to the C1 carbonic acid site on NEPP6 by using achemical linker. With streptavidin as a probe, we could then detectproteins bound to NEPP6-biotin. In order to study thiols as targets ofNEPP binding, BSA (1 μg per lane) was incubated with variousconcentrations (0-1000 μM) of N-ethylmaleimide (NEM) for 30 minutes atroom temperature. Vehicle or NEPP6-biotin (10 μM) in phosphate-bufferedsaline (PBS) was added and the mixture was incubated at room temperaturefor 5 h, subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and probed with peroxidase-conjugatedstreptavidin. The gel was also stained with Coomassie brilliant blue.After exposure to NEPP6-biotin, a single band (68 kDa) corresponding toBSA/NEPP6-biotin was detected. Pretreatment with N-ethylmaleimide (NEM)depressed this signal in a dose-dependent manner, although levels ofprotein were virtually the same as judged from Coomassie brilliant bluestaining of the gel. NEM also abolished the binding of NEPP6-biotin tolysates of HT22 cells or brain. These results suggest that cysteinethiols are the target of NEPP compounds binding to cellular proteins.

NEPPs Activate HO-1 Transcription Through the ARE.

We next studied activation of the HO-1 promoter and ARE by NEPP. Inorder to examine the induction of HO-1 protein by NEPP11, variousconcentrations of NEPP11 were added to HT22 cells for 24 h. Thereafter,cell lysates (10 μg per lane) were subjected to SDS-PAGE and probed withanti-HO-1 or anti-β-actin. We found that NEPP11 induced HO-1 proteinlevels approximately five-fold over baseline conditions at the same NEPPconcentration that prevented neuronal cell death (Satoh et al., Eur. J.Neurosci. 17:2249-2255, 2003). Next, we studied transcriptionalactivation of the HO-1 gene by NEPP11 in HT22 cells, a differentiatedneuronal cell line, transfected with pHO15luc, a luciferase reporterconstruct under the control of a 15-kb mouse HO-1 promoter fragment.HT22 cells were transfected with reporter cDNAs (1 μg per well), andvarious concentrations of NEPP11 were added to the cultures. After a24-h incubation, cell lysates were subjected to luciferase reporterassays. Based on luciferase activity, NEPP11 stimulated the expressionof HO-1 transcription more than five-fold in a dose-dependent manner.Similar responses were obtained with NEPP6. Gong et al. (Antioxid. RedoxSignal. 4:249-257, 2002) had identified two enhancer regions, E1 and E2,located upstream of the transcriptin initiation site by approximately 4and 10 kb that contained the elements responsible for activation by15-deoxy-Δ^(12,14)-PGJ₂ (Gong et al., Antioxid. Redox Signal. 4:249-257,2002). To determine the role of the E1 and E2 enhancers, we expressed amutant promoter construct lacking both enhancer sites[pHO15lucΔ(E1+E2)]. The mutant was only minimally responsive to NEPP11(1.8-fold induction). Similar results were obtained with NEPP6.

The E1 and E2 enhancer sites in the HO-1 promoter each contain an ARE.Recent evidence has suggested that electrophiles can activate the AREvia the Keap1/Nrf2 pathway (Itoh et al., Mol. Cell. Biol. 24:36-45,2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002). BecauseNEPP compounds have an electrophilic carbon at position #11 and can bindcysteine residues of cellular proteins, we studied this pathway of AREactivation. To provide direct evidence that NEPP11 could activate theHO-1 enhancer through an ARE, we studied transcriptional activation of awild-type ARE core element (pAREluc) and a mutated form (pGC-AREluc)(Lee et al., Biochem, Biophys. Res. Commun. 280:286-292, 2001). cDNAconstructs represented wild-type pAREluc(5′-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAG-CAGAATC-3′) and mutant pGC-AREluc(5′-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAATAGAATC-3′) (Kraft et al., J.Neurosci. 24:1101-1112, 2004). NEPP11 stimulated the wild-typetranscriptional activity up to seven-fold in a dose-dependent manner,whereas the mutant was unaffected. Similar responses were obtained withNEPP6. These results strongly support the notion that NEPP compoundsactivate the HO-1 enhancer by activating an ARE.

Because the E1 and E2 regions also contain other enhancer elements, wetested the effects of NEPP11 (2 μM) on the expression of luciferaseactivity derived from these elements, including the cAMP-responsiveelement (CRE; p3xCREluc), AP-1 binding site (p3xAP1luc), NF-κB bindingsite (p3xNF-κBluc), NFAT-binding site (p3xNFATluc), ETS-binding site(p3xETSluc), and MEF2-binding site (p3xMEF2luc). The plasmid pHO15luc,used for comparison, showed greater than five-fold induction of reporteractivity. In contrast, NEPP11 (2 μM) had no effect on the activity ofp3xMEF2luc, slightly depressed the activities of the p3xCREluc,p3xAP1luc, p3xNFATluc, p3xETSluc constructs, and minimally activatedp3xNF-κBluc expression. These results suggest that activation of thesetranscriptional elements plays a minor role in the activation of theHO-1 promoter by NEPP11.

To confirm that NEPP11 leads to an increase in transcription factors,including Nrf2, that bind to the ARE, we performed EMSAs. HT22 cellswere incubated for 8 h with vehicle or NEPP11 (1 μM). EMSAs wereperformed by using 10 μg of nuclear lysates per lane and abiotin-labeled ARE probe. In one lane, anti-Nrf2 antibody (100×) wasadded to supershift Nrf2 protein binding to ARE probe. In another lane,an excess amount of non-labeled probe was added to the sample to competeout the labeled ARE probe from binding to proteins in the nuclearlysates. The band representing the labeled ARE probe was shifted in thepresence of control cell lysates. The band representing the labeled AREprobe was shifted in the presence of control lysates, indicating bindingof endogenous transcription factors to the ARE. Lysates prepared afterexposure to NEPP11 (1 μM) manifest a significant increase in theintensity of this band. In contrast, the band was totally abrogated inthe presence of excess unlabeled probe. Importantly, addition ofanti-Nrf2 antibody produced a supershifted band, consistent with thenotion that one of the transcription factors binding to the ARE underthese conditions represented Nrf2.

Activation of the Keap1/Nrf2 Pathway by NEPP Compounds in a NeuronalCell Line.

Next, we examined the localization of an Nrf2-Green Fluorescent Protein(GFP) fusion protein (Numazawa et al., Am. J. Physiol. 285:C334-C342,2003) in transfected HT22 cells. HT22 cells transfected with pNrf2-GFPwere treated with vehicle or NEPP11 (2 μM) for 24 h and observed underepifluorescence microscopy. Under basal conditions, the Nrf2-GFP fusionprotein was predominantly localized in the cytoplasm but translocatedinto the nucleus upon exposure to 2 μM NEPP11. In general, Nrf2 israpidly ubiquitinated and degraded by the proteasome pathway in thecytoplasm but becomes stable when translocated into the nucleus (Itoh etal., Mol. Cell. Biol. 24:36-45, 2004; Gong et al., Antioxid. RedoxSignal 4:249-257, 2002). Thus we hypothesized that nuclear levels ofNrf2 protein should increase when cells are exposed to an electrophilesuch as NEPP, which promotes nuclear translocation. To test thishypothesis, cytosolic and nuclear fractions (200 μg protein) from cellsthat had been treated with vehicle or 2 μM NEPP11 for 24 h wereprecipitated and probed with anti-Nrf2 antibody and with anti-NeuN, aneuronal-specific nuclear protein. Indeed, we confirmed this hypothesis.

To elucidate the mechanism of electrophile action in this regard, weexamined whether NEPP compounds bind Keap1. To examine induction of HO-1protein by NEPP6-biotin, lysates (10 μg per lane) of HT22 cells treatedwith various concentrations of NEPP5-biotin were probed with anti-HO-1or with anti-β-actin. Similar to NEPP6 and -11, we found that theconjugated product of NEPP6 and biotin (NEPP6-biotin) is neuroprotective(Satoh et al., J. Neurochem. 77:50-62, 2001). Additionally, atconcentrations of 1-10 μM, NEPP6-biotin induced the expression of HO-1protein, confirming that NEPP6-biotin retains its biological effect. Wethen performed immunoprecipitation experiments to provide directevidence for adduct formation between NEPP compounds and Keap1. HT22cells were treated with vehicle or 10 μM NEPP6-biotin, incubated for 24h. Subsequently, lysates were prepared and subjected to precipitationwith either anti-Keap1 or streptavidin. The precipitates wereelectrophoresed and probed. A 73-kDa protein, corresponding to Keap1that had bound to NEPP6-biotin, was observed in cells treated withNEPP6-biotin but not with vehicle. The precipitates were also probedwith anti-Keap1 to confirm that the amount of precipitated Keap1 was thesame between vehicle- and NEPP6-biotin-treated cells. Next, the lysateswere treated in reverse fashion, i.e., precipitated with streptavidinand then probed with anti-Keap1 antibody. Again, precipitated protein,corresponding to Keap1 that had bound to NEPP6-biotin, was detected onlyin the cells treated with NEPP6-biotin, but not with vehicle. Takentogether, these results are consistent with the notion that NEPPcompounds bind to Keap1 in cells.

To demonstrate that binding of NEPP compounds to Keap1 protein isinvolved in the biological actions of NEPPs, we examined the effects ofa Keap1 cysteine mutant (C151S, in which cysteine residue #151 has beenreported to be essential for activation of Nrf2-mediated transcriptionby electrophiles (Zhang and Hannink, Mol. Cell. Biol. 23:8137-8151,2003). Overexpression of this mutant should abolish the functional linkbetween Keap1 and Nrf2 proteins and, thus, reduce sensitivity toelectrophiles, such as NEPP11. Along these lines, we found thatactivation of the HO-1 promoter (pHO15luc) by NEPP11 was significantlydepressed by co-transfection of pKeap1(C151S), but not wild-type pKeap1.

Activation of the Keap1/Nrf2 Pathway in Primary Cortical Cultures.

To begin to test the effects of NEPP compounds on primarycerebrocortical neurons, we treated mixed neuronal/glial corticalcultures with NEPP6-biotin and then fixed and stained with rhodamineconjugated to streptavidin to determine the site of NEPP accumulation.Cortical cultures treated with NEPP6-biotin (10 μM) were stained withanti-MAP-2 monoclonal and rhodamine-conjugated streptavidin antibodiesand with Hoechst 33,258 dye. We found that MAP-2-positive neuronsstained strongly for NEPP6-biotin-streptavidin, suggesting that NEPPcompounds accumulate in neurons. Incubation in biotin by itself did notresult in neuronal accumulation of biotin-streptavidin, and incubationin NEPP6-biotin plus excess free biotin (4 mM) did not affect the degreeof neuronal accumulation, indicating that NEPP6-biotin accumulation wasnot facilitated by the presence of biotin but rather by NEPP itself.

We reasoned that if, unlike other electrophiles (Kraft et al., J.Neurosci. 24:1101-1112, 2004), NEPPs accumulate predominantly inneurons, then HO-1 might be induced preferentially in this cell type. Tocheck this possibility, we performed immunofluorescence studies withanti-HO-1 antibody after exposure to a potent NEPP compound. Corticalcultures (E17 and DIV14-21) treated with vehicle or NEPP11 (0.7 μM) werestained with anti-MAP-2 and anti-HO-1 and with Hoechst dye. In controlcultures, non-neuronal cells expressed relatively more HO-1 at baselinethan did neurons. After addition of NEPP11, HO-1 immunofluorescenceincreased mainly in neurons, in both the cytosol and nucleus. To examineinduction of HO-1 protein after exposure to NEPP, lysates (10 μg perlane) of primary cortical cultures treated with vehicle or NEPP11 for 24h were probed with anti-HO-1 or anti-β-actin in immunoblots. NEPP11 alsoincreased total HO-1 protein in the cultures. The fold induction of HO-1by NEPP11 was 2.2±0.25 (for 0.5 μM NEPP11) and 3.5±0.25 (for 1.0 μMNEPP11 as assessed by densitometry.

Next, transcriptional activation of the HO-1 promoter by NEPP wasexamined by reporter gene assays in primary neurons. Cortical cultureswere transfected with reporter cDNAs (1 μg per well), and 0.7 μM NEPP11was added to the cultures. After a 24 h incubation, cell lysates weresubjected to luciferase reporter assays. In these transfected corticalcultures, NEPP11 (0.7 μM) significantly increased activity of the HO-1promoter and ARE core element, an effect that was abrogated my mutationof the HO-1 enhancer sites or the ARE site, respectively. These resultssuggest that NEPP11 induced HO-1 protein in primary cortical neuronsthrough activation of the ARE elements in the HO-1 promoter. If theKeap1/Nrf2 pathway, indeed, mediates activation of the HO-1 promoter byNEPP11, mutant Nrf2 protein should inhibit this activation. For thispurpose, we used the mutated construct pNrf2(S40A)-GFP (in which theserine residue at position #40 is replaced by an alanine); the encodedprotein does not activate the ARE, because it cannot translocated intothe nucleus (Numazawa et al., Am. J. Physiol. 285:C334-C342, 2003).NEPP11 significantly activated the HO-1 promoter, and cotransfectionwith pNrf-GFP did not affect activation. In contrast, cotransfectionwith pNr12(S40A)-GFP almost completely knocked down activation by NEPP11(additionally, the basal level of HO-1 promoter activity was reduced).Taken together, our results suggest that NEPP11 activated the Keap1/Nrf2pathway selectively in neurons. Moreover, the selective activation inneurons may explain the relatively small amplitude of total HO-1activation seen in the mixed neuronal/glial culture system in which gliapredominate.

Neuroprotection by NEPP11.

Neuron-selective activation of the Keap1/Nrf2/Ho-1 pathway by NEPPcompounds should provide neuroprotection. Hence, we examined the actionof NEPP11 both in vitro and in vivo in excitotoxic paradigms, first inculture as a protectant from NMDA-receptor-mediated insults and thenafter middle cerebral artery occlusion (MCAO) by using the intraluminalfilament model of transient focal ischemia/reperfusion in mice.

Exposure of primary cortical cultures to relatively mild insults, suchas low concentrations of NMDA (50 μM) for short durations (15 min) isknown to cause delayed and predominantly apoptotic neuronal cell death(Bonfoco et al., Proc. Natl. Acad. Sci. USA 92:7162-7166, 1995). Westained the cultures with both anti-MAP-2 and anti-NeuN monoclonalantibodies to label neuronal dendrites and nuclei, respectively. Vehicleor NEPP11 (0.7 μM) was added to cerebrocortical cultures (E17 andDIV14-21) 60 min before treatment with NMDA (50 μM) for 15 min. Thecultures were then incubated for 20 h and subsequently stained withanti-MAP-2 and anti-NeuN and with Hoechst dye. Apoptotic nuclei wereidentified by morphological changes seen with Hoechst staining. In thissystem, NEPP11 significantly decreased the number of apoptotic neurons,suggesting that NEPP11 protected neurons against excitotoxicity invitro. We also added zinc protoporphyrin (ZnPP, 10 μM), a relativelyspecific HO-1 antagonist, simultaneously with NEPP11. The number ofapoptotic neurons was assessed by determining the apoptotic index[(number of condensed nuclei in MAP-2 or NeuN-positive cells)/(number oftotal MAP-2 or NeuN-positive cells)×100%], as reported by Bonfoco et al.(Proc. Natl. Acad. Sci. USA 92:7162-7166, 1995). ZnPP abrogated theneuroprotective effect of NEPP11 in these cerebrocortical cultures. Thisresult is consistent with the notion that NEPP11 protects primarycortical neurons against excitotoxicity, at least in part, throughinduction of HO-1. If this antioxidant pathway is important for NEPPaction, then downstream events should also be affected. Along theselines, we found that NEPP11 (1 μM) inhibited NMDA-induced caspase-3activation. In contrast, if this is the predominant pathway, then otherknown anti-apoptotic genes, e.g., bcl-xL and bcl-2, might not be inducedby NEPP compounds. Indeed, we found this to be the case.

One caveat to the mechanism of NEPP and other electrophilic compoundsacting at the level of Keap1 to induce HO-1 transcription is that NEPPcould potentially react indiscriminately with other thiol-containingcompounds in cells. To approach this question, we assessed cellularglutathione (GSH) levels to determine whether NEPP11 would affect thisabundant antioxidant thiol. Nepp11 or N-ethylmaleimide (NEM) was addedat t=0 and levels of total GSH were measured at the indicated times. GSHcontent of control cortical cultures (set arbitrarily at 100%) was46.8±4.5 nmol/mg protein. Unlike NEPP11, NEM did not produceneuroprotection in these cultures and, in fact, resulted in neuronaldeath after 24 h. NEPP11 did not deplete GSH levels in corticalcultures, unlike many other electrophiles that have this effect (e.g.,NEM). In fact, GSH levels transiently increased after exposure toNEPP11. This increase in GSH may have occurred by induction of γ-GCL(Sagara et al., J. Biol. Chem. 277:36204-36215, 2002), the rate-limitingenzyme in GSH biosynthesis, and could also contribute to cytoprotection.In fact, this scenario seems likely, because the Keap1/Nrf2 pathwayregulates the expression of γ-GCL in addition to HO-1 (Itoh et al., Mol.Cell. Biol. 24:36-45, 2004). Besides GSH, cells have another majorreduction pathway representing the thioredoxin-glutaredoxin system.However, we found that NEPP11 (1 μM) did not significantly affect theexpression of thioredoxin or glutaredoxin under our conditions (Example2).

Next, we tested whether NEPP11 could decrease the size of brain infarctsafter MCAO/reperfusion injury. NEPP11 or vehicle was injectedintraperitoneally (i.p.) 1 h before and 4 h after MCAO. The area ofbrain infarction (corrected for possible edema) was assessed on coronalsections from vehicle- and NEPP11-treated mice stained with 2.5%2,3,5-triphenyltetrazolium chloride 24 h after the onset of reperfusion.We monitored physiological variables, including arterial pressure, bloodgases and glucose, core body temperature, and regional cerebral bloodflow; these parameters did not differ between the control andNEPP11-treated groups. NEPP11 significantly reduced the infarct area incoronal sections, suggesting that NEPP11 is neuroprotective in vivo. Wedid not examine the effects of NEPP11 administered post-infarct in thisstudy, because the drug requires several hours to exert itsneuroprotective effect by transcriptional activation and thereforerequires pre-treatment (Satoh et al., Eur. J. Neurosci. 17:2249-2255,2003; Satoh et al., J. Neurochem. 77:50-62, 2001).

To test the hypothesis that NEPP protection against brain ischemia isassociated with HO-1 expression, we examined HO-1 induction by Westernblotting and immunostaining. NEPP11 or vehicle was injectedintraperitoneally (i.p.) 12 h before the animals were killed. Brainlysates (10 μg per lane) were extracted and subjected to Westernblotting with anti-HO-1 and anti-β-actin antibody. For immunostaining,coronal sections of mouse brain were stained with anti-MAP-2 andanti-HO-1 antibodies. We found that the same concentration of NEPP11that prevented neuronal cell death during brain ischemia increased thelevel of HO-1 protein in the brain. Induction of HO-1 protein wasobserved in neuronal soma and dendrites.

Discussion.

This study provides evidence that electrophilic drugs can affordneuroprotection through activation of the Keap1/Nrf2 pathway andconsequent up-regulation of HO-1 and possibly other class II enzymes. Itwas known that up-regulation of HO-1 decreased stroke size, as assessedin HO-1 transgenic mice (Maines and Panahian, in Hypoxia: From Genes tothe Bedside, eds. Roach et al. (New York: Kluwer), 2001, pp. 249-272).Here, we develop and characterize a set of small-molecule electrophilesthat activate HO-1 transcription in neurons, showing that this pathwayrepresents a druggable target in the brain. Successful neuroprotectionby NEPP compounds involves activation of the Keap1/Nrf2 pathway atnontoxic concentrations. Many other electrophilic molecules causesystemic side effects and are not neuroprotective, probably because theyalso deplete critical reducing substances in the cell, such as GSH, butthis is not the case with the NEPP drugs.

NEPP compounds are lipophilic, an important characteristic for theiraccumulation in neurons, as demonstrated in this study with labeled NEPP(NEPP-biotin). Kraft et al. (J. Neurosci. 24:1101-1112, 2004), however,reported that another electrophile, tert-butylhydroquinone (TBHQ),activates the ARE in astrocytes, a fact that may appear inconsistentwith our observations. Nevertheless, it should be noted that thechemical structures of electrophiles such as NEPP and TBHQ vary widelyand may affect their cellular uptake (Kraft et al., J. Neurosci.24:1101-1112, 2004). Hence, one electrophile may very well localize toastrocytes, whereas another predominates in neurons, as observed herefor NEPP compounds. NEPP compounds (Δ⁷-prostaglandinA₁ analogues) havebeen molecularly designed based on the chemical structure ofΔ¹²-prostaglandinJ₂, and these molecules share many chemical andbiological properties (Fukushima, Eicosanoids 3:189-199, 1990).Δ¹²-prostaglandinJ₂ is reportedly transported into cells by activetransport through the cell membrane (Narumiya et al., J. Pharmacol. Exp.Ther. 239:506-511, 1986). Therefore, neurons may have a more activetransport system for NEPP compounds than do glia, because we observedthat NEPP compounds accumulate preferentially in neurons. In contrast,the electrophile TBHQ may simply diffuse into cells and, thus, affectglia, which greatly outnumber neurons.

Our findings suggest the neuroprotective mechanism of NEPP action.Within cells, these drugs bind to the cytosolic regulator protein Keap1,which, in turn, liberates Nrf2. Nrf2 is then translocated into thenucleus, where it activates AREs on the HO-1 promoter (Itoh et al., Mol.Cell. Biol. 24:36-45, 2004; Gong et al., Antioxid. Redox Signal.4:249-257, 2002). Transcription of HO-1 is thus activated in neurons,and an increase in HO-1 protein leads to degradation of heme molecules,producing biliverdin and bilirubin (Itoh et al., Mol. Cell. Biol.24:36-45, 2004; Gong et al., Antioxid. Redox Signal. 4:249-257, 2002).The accumulation of bilirubin, a potent antioxidant molecule, isresponsible, at least in part, for the neuroprotective effects of HO-1,and thus of NEPP compounds (Satoh et al., Eur. J. Neurosci.17:2249-2255, 2003; Sagara et al., J. Biol. Chem. 277:36204-36215,2002). Additionally, we found that inhibition of HO-1 by zincprotoporphyrin prevented the protective effect of NEPP, consistent withthe notion that the therapeutic action of these drugs is mediatedpredominantly by this pathway.

Recently, decreased Nrf2 transcriptionally activity was also reported tocause age-related loss of GSH synthesis (Suh et al., Proc. Natl. Acad.Sci. USA 101:3381-3386, 2004). Low molecular-weight compounds can induceγ-GCL through activation of the ARE to increase GSH levels. Thus,compounds that regulate the Keap1/Nrf2 pathway may be promisingcandidates for neuroprotection against free radical stress throughinduction of γ-GCL as well as HO-1, both of which help preventaccumulation of reactive oxygen species.

In summary, we found that modulation of the Keap1-Nrf2 pathway by NEPPcompounds leads to activation of the HO-1 promoter by Nrf2. Induction ofHO-1 protein is known to play an important neuroprotective role againstexcitotoxicity and brain ischemia (Maines and Panahian, in Hypoxia: FromGenes to the Bedside, eds. Roach et al. (New York: Kluwer), 2001, pp.249-272; Stocker et al., Science 235:1043-1046, 1987; Dore et al., Proc.Natl. Acad. Sci. USA 96:2445-2450, 1999; Poss and Tanegawa, Proc. Natl.Acad. Sci. USA 94:10925-10930, 1997; Satoh et al., Eur. J. Neurosci.17:2249-2255, 2003; Satoh et al., J. Neurochem. 77:50-62, 2001). How canclinically useful drugs be developed based on the chemical structures ofcyclopentenone prostaglandins like the NEPPs? One approach is tosynthesize neuroprotective electrophilic drugs like the NEPPs that spareessential redox elements. Strongly electrophilic compounds are known todeplete the cell of critical thiol-containing compounds like GSH and,hence, contribute to cell death. In contrast, NEPP compounds and theircongeners interact with Keap1 without depleting GSH. Selectiveactivators of the Keap1/Nrf2 pathway are neuroprotective agents that actthrough induction of phase 2 genes, including HO-1.

Example 2 Methods and Materials

Focal Cerebral Ischemia and Reperfusion. NEPP11 was injected at 100mg/ml in a 7.5% solution of DMSO in PBS; controls received the diluentalone. The investigator was blinded as to the treatment group. Theinjected volume was 10 ml/g of body weight, corresponding to 1 mg/kg.The intraluminal-filament model of middle cerebral artery occlusion(MCAO)/reperfusion was used (Gu et al., Science 297:1186-1190, 2000).Male mice (C57BL/6) at age 6-8 weeks and weighing 20-30 g were housed ina 12 hr light/12 hr dark cycle and permitted food and water intake adlibitum. The animals were anesthetized with an isoflurane and 70%nitrous oxide/30% oxygen mixture delivered through a nose cone. Coretemperature was maintained at 37±1° C. Other physiological parameterswere monitored, including systemic blood pressure, glucose, and arterialblood gasses and pH. The mice underwent a 2 hr MCAO followed by a 24 hrreperfusion period. Occlusion and reperfusion of blood flow weremonitored by laser Doppler flowmetry. Anesthesia was maintained for theduration of the surgical procedure, which typically lasted 10 min in ourhands.

After the reperfusion period, the animals were killed. The brains werethen sliced into sections of 2 mm thickness. Each slice was incubatedfor 10 min in a 2.5% solution of 2,3,5-triphenyltetrazolium chloride at37° C. and fixed in 4% buffered formaldehyde solution. Areas ofinfarction occurred in the right middle cerebral artery territory ofeach brain slice and were quantified with a computerized image analysissystem (NIH image 1.62) as described (Gu et al., Science 297:1186-1190,2000).

Cell Cultures, Transfection, and Glutathione (GSH) Measurement. HT22cells were cultured as described (Satoh et al., Eur. J. Neurosci.17:2249-2255, 2003; Satoh et al., J. Neurochem. 77:50-62, 2001; Sagaraet al., J. Biol. Chem. 277:36204-36215, 2002). Cerebrocortical cultureswere prepared from embryonic day-17 Sprague-Dawley rats and used at day14-21 in vitro (DIV14-21), as described (Bonfoco et al., Proc. Natl.Acad. Sci. USA 92:7162-7166, 1995). To induce predominantly neuronalapoptosis (rather than necrosis), we exposed cortical cultures to 50 mMNMDA plus 5 mM glycine/1.8 mM CaCl₂ in nominally Mg²⁺-free Earle'sbalanced salt solution for 15 min (Bonfoco et al., Proc. Natl. Acad.Sci. USA 92:7162-7166, 1995). After exposure to NMDA, the cultures werereturned to normal medium containing vehicle or NEPP11 and incubated for20 hr before analyzing cell survival. To assess the ability of NEPPcompounds to block NMDA-induced neuronal apoptosis, we identifiedapoptotic neurons by double immunofluorescence with anti-NeuN andanti-MAP-2 to specifically label neurons and with Hoechst staining fornuclear morphology to detect apoptosis. The percentage of neurons andnon-neuronal cells in our cultures was 33.6±4.9% and 66.4±4.4% (n=4),respectively.

Transfections were performed with Lipofectamine 2000 (Invitrogen), andfirefly luciferase activity in cell lysates was measured with aluminometer in the reporter gene assays (Promega). Total GSH (reducedand oxidized) was determined as described (Sagara et al., J. Biol. Chem.277:36204-36215, 2002).

Results

Lack of Effect of NEPP Compounds on Expression of Trx and Grx Genes. Theexpression of Trx1/2 and Grx1/2 was examined in cortical culturesincubated with NEPP11 (1 mM) for 24 hr by using RT-PCR with thefollowing primers (Jurado et al., J. Biol. Chem. 278:45546-45554, 2003):Trx1, forward: 5′-CGT GGT GGA CTT CTC TGC TAC GTG GTG-3′; reverse:5′-GGT CGG CAT GCA TTT GAC TTC ACA GTC-3′; Trx2m, forward: 5′-GCT AGAGAA GAT GGT CGC CAA GCA GCA-3′; reverse: 5′-TCC TCG TCC TTG ATC CCC ACAAAC TTG-3′; Grx-1, forward: 5′-TGC AGA AAG ACC CAA GAA ATC CTC AGTCA-3′; reverse: 5′-TGG AGA TTA GAT CAC TGC ATC CGC CTA TG-3′; Grx-2,forward: 5′-CAT CCT GCT CTT ACT GTT CCA TGG CCA A-3′; reverse: 5′-TCATCT TGT GAA GCG CAT CTT GAA ACT GG-3′. We found that NEPP11 (1 mM) didnot significantly affect expression of the Trx and Grx genes under theconditions of our assay.

Example 3

Glutamate, the major excitatory amino acid in the brain, exerts variousactions on neurons, affecting development, plasticity, and survival(Nakanishi, Trends Neurosci. 28:93-100, 2005; Barco et al., J.Neurochem. 97:1520-1533, 2006). Under physiological conditions,glutamate plays a major role in learning and memory in part via NMDAreceptor-mediated pathways (Nakanishi, Trends Neurosci. 28:93-100, 2005;Barco et al., J. Neurochem. 97:1520-1533, 2006). However, underpathological conditions, glutamate can induce neuronal cell death,termed “excitotoxicity,” predominantly by excessive activation of theNMDA receptor (Choi, J. Neurosci. 23:1261-1276, 1992; Ankarcrona et al.,Neuron 15:961-973, 1995; Hara and Snyder, Annu. Rev. Pharmacol. Toxicol.47:117-141, 2007) and in part owing to the subsequent generation of freeradicals such as nitric oxide (NO) and reactive oxygen species (ROS). Inimmature neurons, which have not yet expressed functional NMDAreceptors, high concentrations of glutamate induce a novel type ofneuronal death mediated by depletion of reduced glutathione (GSH),termed “oxidative glutamate toxicity” (Murphy et al., Neuron2:1547-1558, 1989; Murphy et al., FASEB J. 4:1624-1633, 1990; Darguschand Schubert, J. Neurochem. 81:1394-1400, 2002). Although these twotypes of neuronal death induced by glutamate are distinct from eachother, oxidative stress is involved in both types (Murphy et al., Neuron2:1547-1558, 1989; Coyle and Puttfarcken, Science 262:689-695, 1993;Dugan et al., J. Neurosci. 15:6377-6388, 1995). For this reason, severalantioxidant molecules have been reported to protect neurons against bothexcitotoxicity and oxidative glutamate toxicity (Murphy et al., FASEB J.4:1624-1633, 1990; Ankarcrona et al., Neuron 15:961-973, 1995; Darguschand Schubert, J. Neurochem. 81:1394-1400, 2002). Thus, one strategy forthe development of neuroprotective drugs is to search forlow-molecular-weight compounds that can regulate redox state and therebycounter oxidative damage (Satoh and Lipton, J. Neurochem. 75:1092-1102,2007).

Recently, our group, in addition to those of Johnson and Murphy,reported that a series of compounds not necessarily possessingantioxidative activity themselves could nonetheless transcriptionallyinduce antioxidative enzymes to afford neuroprotection (Satoh et al.,Proc. Nat. Acad. Sci. USA 103:768-773, 2006; Kraft et al., J. Neurosci.24:1101-1112, 2004; Shih et al., J. Neurosci. 25:10321-10335, 2005).Such electrophilic compounds have an advantage over antioxidantmolecules because their action is more sustained and amplified bytranscription-mediated signaling pathways (Satoh and Lipton, TrendsNeurosci. 30:38-45, 2007). Electrophiles induce the expression of a setof antioxidant enzymes, called “phase 2 enzymes,” including hemeoxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), andγ-glutamyl cysteine ligase (γ-GCL), all of which provide efficientcytoprotection by regulating the intracellular redox state (Talalay,Biofactors 12:5-11, 2000; Padmanabhan et al., Mol. Cell 21:689-700,2006; Itoh et al., Free Radic. Biol. Med. 36:1208-1213, 2004).Representing a specific transcriptional element located in the 5′upstream promoter region of genes that encode phase 2 enzymes, theantioxidant-responsive element (ARE) plays a central role in theinduction of such enzymes (Talalay, Biofactors 12:5-11, 2000;Padmanabhan et al., Mol. Cell 21:689-700, 2006; Itoh et al., Free Radic.Biol. Med. 36:1208-1213, 2004). A key cascade involved is termed theKeap1/Nrf2 pathway, which is comprised of Keap1, a regulator protein,and Nrf2, a transcription factor that binds to the ARE. Keap1 is anadapter protein that facilitates ubiquitination of Nrf2 and thus drivesconstitutive degradation of this transcription factor. Whenelectrophiles react with critical cysteine residues on the Keap1 proteinto form an adduct, they perturb this system, thereby stabilizing Nrf2and allowing it to be translocated from the cytoplasm into the nucleus,where it binds to AREs and stimulates the transcription of phase 2 genes(Talalay, Biofactors 12:5-11, 2000; Padmanabhan et al., Mol. Cell21:689-700, 2006; Itoh et al., Free Radic. Biol. Med. 36:1208-1213,2004).

Neuroprotective electrophilic compounds reported previously may bedivided into two major groups, catechol- and enone-types, as shown inFIG. 2 (Kraft et al., J. Neurosci. 24:1101-1112, 2004; Satoh et al.,Proc. Nat. Acad. Sci. USA 103:768-773, 2006; Satoh and Lipton, TrendsNeurosci. 30:38-45, 2007). These two types of compounds manifestdistinctive features. One difference lies in their degree ofelectrophilicity. Enone-type electrophilic compounds, including theenone-type curcumin (Yazawa et al., FEBS Lett. 580:6623-6628, 2006) anddienone-type NEPP11 (Satoh et al., Proc. Nat. Acad. Sci. USA103:768-773, 2006), are themselves electrophilic. In contrast,catechol-type compounds are not themselves electrophilic but becomeelectrophilic by oxidative conversion to a quinone (Nakamura et al.,Biochem. 15:4300-4309, 2003). Thus, these catechol-type compoundsfunction as prodrugs, which require conversion from catechol to quinoneto exert their neuroprotective effect (Satoh and Lipton, TrendsNeurosci. 30:38-45, 2007). Another distinctive difference between thecatechol-type and quinone-type is their distribution in neuronalcultures. Tert-butyl hydroquinone (TBHQ), a catechol-typeneuroprotective electrophilic compound, reportedly acts preferentiallyin astrocytes and protects neurons by a paracrine mechanism(Ahlgren-Beckendorf et al., Glia 15:131-142, 1999; Lee et al., J. Biol.Chem. 278:12029-12038, 2003; Kraft et al., J. Neurosci. 24:1101-1112,2004). In contrast, NEPP11, an enone-type neuroprotective electrophiliccompound, accumulates in neurons to induce HO-1, thereby exerting adirect protective action on neurons (Satoh et al., Proc. Nat. Acad. Sci.USA 103:768-773, 2006).

Since plants produce a variety of electrophilic compounds, we looked fornaturally-occurring electrophilic compounds in plants of thecatechol-type that might protect neurons through transcriptionalactivation. Carnosic acid is a naturally-occurring catechol-typepoly-phenolic diterpene obtained from Rosmarinus officinalis (rosemary)and comprises about 5% of the dry weight of rosemary leaves (Kosaka andYokoi, Bio. Pharm. Bull. 26:1620-1622, 2003). CA reportedly has variousbiological actions, possibly effected through phosphatidylinositol3-kinase (Martin et al., J. Biol. Chem. 279:8919-8929, 2004), peroxisomeproliferator-activated receptor (PPAR). (Rau et al., Planta Med.72:881-887, 2006), cyclin A/B1 (Visanji et al., Cancer Lett.237:130-136, 2006), and free radical-scavenging activity (Aruoma et al.,Xenobiotica 22:257-268, 1992).

Electrophilic compounds are a newly-recognized class of redox-activeneuroprotective compounds with electron deficient, electrophilic carboncenters that react with specific cysteine residues on targeted proteinsvia thiol (S-) alkylation. Although plants produce a variety ofphysiologically-active electrophilic compounds, the detailed mechanismof action of these compounds remains unknown. Catechol ring-containingcompounds have attracted attention because they become electrophilicquinones upon oxidation, although they are not themselves electrophilic.We found that CA activates the Keap1/Nrf2 transcriptional pathway bybinding to specific Keap1 cysteine residues, thus protecting neuronsfrom oxidative stress and excitotoxicity. In cerebrocortical cultures,CA-biotin accumulates in non-neuronal cells at low concentrations and inneurons at higher concentrations. Both the neuronal and non-neuronaldistribution of CA may contribute to its neuroprotective effect.Furthermore, CA translocates into the brain, increases the level ofreduced glutathione in vivo, and protects the brain against middlecerebral artery ischemia/reperfusion.

Materials and Methods

Chemicals.

Fraction V bovine serum albumin (BSA), 4′,6-diamino-2-phenylindole(DAPI), dimethyl sulfoxide (DMSO), fluorescein diacetate (FDA),glutamate, Hoechst 33,258, reduced glutathione (GSH), GSSG, oxidizedglutathione (GSSG), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-ethylmaleimide (NEM), N-methyl-D-aspartate(NMDA), propidium iodide (PI), and rotenone, 2,3,5-triphenyltetrazoliumchloride (TTC) were purchased from WAKO Chemicals Inc. (Tokyo, Japan) orSigma (St. Louis, Mo.).

CA and CA-Biotin (CAB).

CA was extracted from rosemary leaves as previously described (Kosakaand Yokoi, Biol. Pharm. Bull. 26:1620-1622, 2003). CA-biotin (CAB) wassynthesized according to Kosaka and Yokoi (Biol. Pharm. Bull.26:1620-1622, 2003) by the following method: Carnosic acid (90 mg),acetonitrile (2 ml), and dicyclohexylcarbodiimide (67 mg; Tokyo KaseiKyogyo Co., Tokyo, Japan) were mixed for three min on ice. Then,5-(biotinamido)pentylamine (41 mg; Pierce, Rockford, Ill.), dissolved in80% acetonitrile, was added to the mixture, which was subsequentlyincubated for 10 min on ice. Thereafter, hydrochloric acid was added toterminate the reaction. The reaction product was separated bypreparative liquid chromatography (ODS column consisting of ODS-S-50c(Maruzen Co., Tokyo, Japan) with acetonitrile and hydrochloric acid assolvent. Both CA and CAB were prepared as 10 mM stock solutions in DMSO.

Western Blotting for CAB.

BSA with or without CAB was separated by 10% SDS-polyacrylamide gelelectrophoresis and then electrophoretically transferred to anitrocellulose membrane (Amersham Life Science, Piscataway, N.J.). Next,the membrane was blocked for 1 h at room temperature in Ca²⁺,Mg2⁺(−)-phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) and5% non-fat dry milk and then incubated for 1 h with horseradishperoxidase-conjugated streptavidin. After the membrane had been washed 3times with PBS-T, signals were detected using ECL Western blottingdetection reagents (Amersham Pharmacia Biotech, Piscataway, N.J.).

Antibodies.

Anti-Nrf2 and anti-Keap1 monoclonal mouse IgGs were generated by KenItoh (Hirosaki University). Other antibodies used were FITC-conjugatedanti-mouse IgG, rhodamine-conjugated anti-mouse IgG,rhodamine-conjugated anti-rabbit IgG, peroxidase-conjugated anti-rabbitIgG, rhodamine-conjugated streptavidin (Jackson Immuno ResearchLaboratories, Westgrove, Pa.), anti-S100β monoclonal antibody,peroxidase-conjugated streptavidin, streptavidin immobilized on4%-beaded agarose (Pierce, Rockford, Ill.), anti-HA monoclonal antibody,and anti-MAP-2, and anti-NeuN monoclonal mouse IgGs, and protein Aimmobilized on Sepharose CL-4B (Sigma, St Louis, Mo.).

Plasmid Constructs.

(1) pGL3-GSTYa ARE Core-Luciferase Vector.

We used single-stranded oligonucleotide containing the GSTYa ARE coresequence (Wasserman and Fahl, Proc. Nat. Acad. Sci. USA 94:5361-5366,1997): sense, 5′-CGC GTT AGC TTG GAA ATG ACA TTG CTA ATG GTG ACA AAG CAACTT TA-3′, and antisense, 5′-GAT CTA AAG TTG CTT TGT CAC CAT TAG CAA TGTCAT TTC CAA GCT AA-3′. The dsDNA thus obtained was inserted into the MluI and Bgl II sites of the pGL3-promoter vector (Promega Co., Madison,Wis.).

(2) pEF6-Nrf2 [Wild-Type (Nrf2WT)], pEF6-Nrf2 [Dominant-Negative(Nrf2DN)], and pEF6-Keap1 Vectors.

Nrf2WT, and Nrf2DN were generated by polymerase chain reaction (PCR)amplification of mouse cDNA with the following oligonucleotides: sensefor Nrf2WT 5′-GCC ATG ATG GAC TTG GAG TTG CCA CCG CCA-3′, sense forNrf2DN 5′-GCC ATG GGT GAA TCC CAA TGT GAA AAT ACA-3′, and commonantisense 5′-GTT TTT CTT TGT ATC TGG CTT CTT GCT TTT-3′ (Alam et al., J.Biol. Chem. 274:26071-26077, 1999). To obtain a Keap1 expression vector,we generated Keap1 cDNA using sense 5′-CCA CCA TGC AGC CCG AAC CCA AGCTTA GC-3′ and antisense 5′-AAG CAA ATT GAT CAA CAA AAC TGT ACC TGC-3′.The amplification products were cloned into the pEF6 vector (Invitrogen,Carlsbad, Calif.).

(3) Expression Vectors for HA-Tagged Keap1 and Keap1 Deletion Mutants.

These constructs were obtained from Dr. Akira Kobayashi of TohokuUniversity (Hosoya et al., J. Biol. Chem. 29:27244-27250, 2006;Kobayashi et al., Mol. Cell. Biol. 26:221-229, 2006).

PC12h and COST Cell Cultures.

Cell lines were cultured and analyzed by cell death assays as previouslydescribed (Satoh et al., J. Neurochem. 75:1092-1102, 2000; Satoh et al.,J. Neurochem. 77:50-62, 2001; Satoh et al., Eur. J. Neurosci.17:2249-2255, 2003). For generation of stable transformants, PC12h cellswere seeded onto 100-mm petri dishes in Dulbecco's Modified Eagle mediumsupplemented with 8% fetal calf serum and 8% horse serum (Invitrogen,Carlsbad, Calif.). The cells were then transfected with pEF6-Nrf2WT orpEF6-Nrf2DN using TransFast (Promega Co., Madison, Wis.). The followingday, the medium was replaced with fresh medium containing 30 μg/mlblastcidine. After two weeks, five colonies of each type of transfectantwere isolated. We screened for high (PC12hW1B) and low(PC12hD5D)-expressing γ-GCL clones by RT-PCR. We found that ARE activitywas high in PC12hW1B and low in PC12hD5D cells, suggesting that thelevels of γ-GCL and ARE activity were well correlated. Expression of theNrf2WT and Nrf2DN constructs was confirmed by RT-PCR

RT-PCR.

In order to examine phase 2 gene induction by CA in PC12h cells andbrain lysates, RT-PCR of total RNA from cortical cultures was performedusing the following primer pairs (Hosoya et al., J. Biol. Chem.29:27244-27250, 2005; Kobayashi et al., Mol. Cell. Biol. 26:221-229,2006): CypA (89 bp), 5′-ACA GGT CCT GGC ATC TTG TC-3′(sense) and5′-AGCCACTCAGTCTTGGCAGT-3′ (antisense); HO-1 (284 bp), 5′-CAG TCG CCTCCA GAG TTT CC-3′(sense) and 5′-TAC AAG GAG GCC ATC ACCAGC-3′(antisense); GCL-M (280 bp), 5′-CTG CTA AAC TGT TCA TTG TAG G-3′(sense) and 5′-CTA TTG GGT TTT ACC TGT G-3′(antisense); GCL-C (213 bp),5′-GTC TTC AGG TGA ACA TTC CAA GC-3′ (sense) and 5′-TGT TCT TCA GGG GCTCCA GTC-3′(antisense); and NQO1 (212 bp), 5′-GTG TAC AGC ATT GGC CACAC-3′(sense) and 5′-AAA TGA TGG CCC ACA GAA AG-3′ (antisense).

Primary Cortical Cultures and Assays for Two Types of GlutamateToxicity.

Cerebrocortical neurons have been used as an in vitro system in order toinvestigate the cellular mechanism of neuronal death caused byglutamate. The level of expression of functional NMDA receptors is keyfor determining whether excitotoxicity or oxidative glutamate toxicitypredominates. With increasing days in vitro (DIV), the expression levelof these receptors in cortical cultures increases. In these immaturecortical cultures, which do not yet express functional NMDA receptors,oxidative glutamate toxicity is dominant; and high concentrations ofglutamate (2 mM) induce cell death via oxidative stress (Murphy et al.,FASEB J. 4:1624-1633, 1990; Lee et al., J. Biol. Chem. 278:37948-37956,2003). In light of this background information, in the present study, weprepared cerebrocortical cultures from embryonic day 17 (E17)Sprague-Dawley rats and examined them at DIV2 as an experimental systemfor oxidative glutamate toxicity (Murphy et al., FASEB J. 4:1624-1633,1990; Lee et al., J. Biol. Chem. 278:37948-37956, 2003). Rotenone, acomplex I inhibitor of the mitochondrial electron-transport chain, alsoinduces oxidative stress in immature cortical cultures (Murphy et al.,FASEB J. 4:1624-1633, 1990; Lee et al., J. Biol. Chem. 278:37948-37956,2003).

In addition, we used mature cortical cultures (E17, DIV21) to assessexcitotoxicity because functional NMDA receptors are expressed on theseneurons. Exposure of mature cortical cultures to relatively mildinsults, such as low concentrations of NMDA (50 μM) for short durations(15 min) has been shown to cause delayed and predominantly apoptoticneuronal cell death in this system; in contrast, higher concentrationsof NMDA for longer exposure times result in necrosis (Bonfoco et al.,Proc. Nat. Acad. Sci. USA 92:7162-7166, 1995; Budd et al., Proc. Nat.Acad. Sci. USA 97:6161-6166, 2000). To induce predominantly neuronalapoptosis (rather than necrosis), we exposed cortical cultures to 50 μMNMDA for 15 min (plus co-agonist 5 μM glycine in 1.8 mM CaCl₂, nominallyMg²⁺-free Earle's balanced salt solution (EBSS) to preventmagnesium-mediated block of NMDA receptor-operated channels) (Bonfoco etal., Proc. Nat. Acad. Sci. USA 92:7162-7166, 1995). After exposure toNMDA, the cultures were returned to normal medium containing vehicle orCA, and then incubated for 20 h prior to analyzing them for cellsurvival. CA or vehicle was present 1 h before the addition of NMDA andremained throughout the experiments. To assess the ability of CA toblock NMDA-induced neuronal apoptosis, we identified apoptotic neuronsby double immunofluorescence labeling with anti-NeuN and anti-MAP-2 tospecifically label neurons, and with Hoechst staining for nuclearmorphology to detect apoptosis. The percentage of neurons andnon-neuronal cells in these mature cortical cultures (E17, DIV21) was33.6±4.9% and 66.4±4.4%, respectively, as determined by use of specificmarkers (Satoh et al., Proc. Nat. Acad. Sci. USA 103:768-773, 2006). Inthe immature cortical cultures (E17, DIV2), the percentage of neuronalcells was over 90% (Satoh et al., J. Neurochem. 77:50-62, 2001).

Immunocytochemistry.

Cultures were fixed with 3% paraformaldehyde at room temperature for 20min. After three washes in PBS, the cells were permeabilized with 0.3%Triton X-100 for 5 min. After three additional washes in PBS, the cellswere incubated at 4° C. overnight with primary antibodies. They werethen washed three times in PBS containing 0.2% Tween 20 (PBS-T), andnext incubated with secondary antibodies for 1 h at room temperature.Thereafter, the cells were again washed, and their nuclei stained withHoechst 33,258 (5 μg/ml) or with DAPI (1 μg/ml) for 5 min. Stainedpreparations were mounted and examined by epifluorescence microscopy.The following primary antibodies were used: anti-Nrf2 antibody,anti-MAP2 and anti-NeuN antibodies to identify neurons (dendrites andnuclei, respectively), and anti-S100β antibody to identify astrocytes.As secondary antibodies, we used FITC-conjugated anti-mouse IgG,rhodamine-conjugated anti-mouse IgG, or rhodamine-conjugatedstreptavidin.

Transient Transfection and Measurement of Luciferase Activity.

PC12h cells or cerebrocortical cultures (E17, DIV21) were incubated for5 h in EBSS containing 1 μg of a plasmid DNA plus Lipofectamine 2000(Invitrogen, Carlsbad, Calif.). Transfection efficiency was normalizedto β-galactosidase activity expressed by co-transfection with pSV-β-gal(Promega). For reporter gene assays, cells were transfected with 1 μg ofthe reporter construct [ARE(GSTYa)-luciferase] and 0.2 μg pSV-β-gal for1 hour. The cells were then washed in PBS alone and incubated in theculture medium for another 24 h with or without CA. Firefly luciferaseactivity and β-galactosidase activity in cell lysates were measuredusing a Luciferase System and β-Galactosidase Enzyme Assay System,respectively (Promega, Madison, Wis.).

Immunoprecipitation with Agarose-Immobilized Streptavidin or Antibody.

PC12h cells (CAB- or vehicle-treated) were lysed in RIPA buffersupplemented with protease inhibitor cocktail. The lysates werecentrifuged at 15,000 rpm for 10 min at 4° C., after which thesupernatants were incubated with agarose-immobilized streptavidin at 4°C. for 1 h. Alternatively, for immunoprecipitation with anti-Keap1, thecell lysates were incubated with the antibody at 4° C. overnight, afterwhich protein A immobilized on Sepharose CL-4B was added and incubationcontinued at 4° C. for 1 h. Then, the complexes were washed three timeswith rinse buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.1% NP-40, 1mM EDTA, and 0.02% NaN3). SDS sample buffer was added, the mixture wasboiled for 5 min, and the supernatants were subjected to SDS-PAGE.Immunoblotting was then performed as described above.

Translocation of CA into the Brain Detected by High-Performance LiquidChromatography (HPLC).

For experiments testing penetration of drug into the CNS, we used adultmale C57BL/6 mice (Charles River) weighing 22-26 g. The mice were keptin cages with ad libitum access to food and water under standardizedhousing conditions (natural light-dark cycle, temperature of 23±3° C.,relative humidity of 50±10%). After a seven-day adaptation to laboratoryconditions, the animals were randomly assigned to each experimentalgroup (n=10 mice each). Four C57 BL/6 mice, fastened for 18 h, were eachorally administered 3 mg of CA in 0.3 ml olive oil. One and three hafter the injection, under ether anesthesia, serum and brain wereisolated for chemical analysis. CA was extracted from tissue withacetonitrile and ethanol. CA levels were obtained by HPLC (column,μBondasphere C18; temperature, 40 .C; HPLC system, Shimadzu [Kyoto,Japan] LC10Avp; detector, UV 230 nm; running solvent 2% acetic acid oracetonitrile, 30% β isocratic at 1 ml/min).

Measurement of Brain GSH.

After exsanguination via heart puncture under ether anesthesia, mousebrains were removed and quickly frozen in liquid nitrogen. We ruled outGSH as a contaminant from blood vessels by HPLC analysis of bloodcomponents in the brain lysates. Along these lines, we observed a majorunknown peak (retention time 12.5 min, probably representing adegradation product of CA) in the serum of CA-injected mice; however,this peak was not detected in the brain lysates, indicating thatcontamination from the blood did not occur under our conditions.

The frozen brains were lysed with 1% sulfosalicylic acid on ice for 10min, and total glutathione (reduced and oxidized) was determined asdescribed previously (Sagara et al., 2002). Briefly, after the lysateshad been incubated on ice for 10 min, the supernatants were collectedafter centrifugation in Eppendorf microfuge tubes. Upon neutralizationof each supernatant with triethanolamine, the total glutathione (reducedand oxidized) concentration was determined. Pure GSH was used to obtainthe standard curve. For the determination of oxidized GSH, the method ofGriffith using 2-vinylpyridine was used to deplete the reduced GSH.

Focal Cerebral Ischemia And Reperfusion.

CA (1 mg/kg) was injected intraperitoneally in a vehicle of 10% DMSO inPBS (10 μl/g body weight); controls received vehicle alone. Theinvestigator was blinded to the treatment group. The intraluminalfilament model of middle cerebral artery occlusion (MCAO)/reperfusionwas utilized, as previously described (Wang et al., Nat. Med. 4:228-231,1998; Gu et al., Science 297:1186-1190, 2002; Gu et al., J. Neurosci.25:6401-6408, 2005; Satoh et al., Proc. Nat. Acad. Sci. USA 103:768-773,2006). Male C57/BL/6 mice, aged 6-8 weeks and weighing 20-30 g, werehoused in a 12-h light/12-h dark cycle and permitted food and waterintake ad libitum. After an overnight fast, the animals wereanesthetized with an isoflurane and 70% nitrous oxide/30% oxygen mixturedelivered through a nose cone. Anesthesia was maintained for theduration of the surgical procedure, which typically lasted 10 min in ourhands. To ensure successful placement of the intraluminal suture forocclusion and subsequent reperfusion, we monitored regional cerebralblood flow (rCBF) in the area of the right middle cerebral artery in allanimals. A laser Doppler flowmeter (Perimed, North Royalton, Ohio) withthe probe fixed on the skull surface (3 mm lateral to midline and 2 mmposterior to the bregma), located at the distal arterial supply of themiddle cerebral artery, measured rCBF, as described previously (Wang etal., Nat. Med. 4:228-231, 1998; Gu et al., Science 297:1186-1190, 2002;Gu et al., J. Neurosci. 25:6401-6408, 2005). All mice subjected to a 2-hright MCAO met the criteria that the rCBF was reduced to <25% of thebaseline during ischemia and recovered to >50% of the baseline within 2h after the onset of reperfusion. Core temperature was maintained at37±1° C. with a servo-controlled heating blanket. We monitored otherphysiological variables, including arterial blood pressure, blood gasesand glucose, and these parameters did not differ significantly betweenvehicle- and CA-treated mice. The right femoral artery was cannulated tomonitor blood pressure and sample arterial blood gases and glucose.Blood pressure was continually recorded before ischemia, duringischemia, and at reperfusion with a blood-pressure transducer, a bridgeamplifier, and a computerized data acquisition system (MacLabs 8s;ADInstruments, Castle Hill, New South Wales, Australia). Arterial bloodgases and glucose were measured before ischemia and 15 min afterreperfusion with a blood gas and glucose analyzer (Stat Profile Ultra C;Nova Biomedical, Waltham, Mass.).

After mice underwent 2-h MCAO followed by 24-h reperfusion, they weresacrificed and their brains sliced into 1-mm thick sections. Each slicewas incubated for 10 min in a 2.5% solution of TTC at 37° C. and thenfixed in 4% buffered formaldehyde solution for storage. To minimize theeffect of brain edema, infarct volume was determined by subtracting thevolume of the contralateral noninfarcted hemisphere (left) from theipsilateral hemisphere (right). Infarction occurred in the right MCAterritory and was quantified with a computerized image analysis system(NIH image, Version 1.62), as described previously (Wang et al., Nat.Med. 4:228-231, 1998; Gu et al., Science 297:1186-1190, 2002; Gu et al.,J. Neurosci. 25:6401-6408, 2005).

Statistical Analysis.

Each experiment was repeated at least three times in quadruplicate. Dataare presented as mean±SEM. Statistical significance was determined by ananalysis of variance (ANOVA) followed by a post hoc Schéffe's test.

Results

CA Binds to GSH and Protein Thiol.

CA has been proposed to donate protons and electrons to oxygen and otheroxygen radicals, as suggested in the case of TBHQ (Nakamura et al.,Biochem. 15:4300-4309, 2003). Simultaneously, CA is oxidized to aquinone. Using nuclear magnetic resonance (NMR), we concluded thatcarbon (14) of CA is the single target of nucleophilic attack by GSHthiol. Facile oxidation of the catechol ring of CA results in conversionto its quinone derivative. Thiol-containing compounds such as GSH caninduce a nucleophilic attack on the electrophilic carbon and thus form aGS-CA adduct.

Next, we assessed the rate of GS-CA adduct formation. Since the GS-CAadduct is highly stable, oxidation from catechol to quinone has beenproposed to be the rate-limiting step of these chemical reactions.Although the reaction solution contained a molar excess of CA and GSH,the GS-CA adduct appeared very slowly. Even after an 18-h incubation,only 17.5% of the CA had reacted to form the adduct. This resultsuggests that the conversion (oxidation) of the catechol to the quinoneform of CA is a very slow process in the cell-free system.

Next, we assessed the binding of CA to other thiols, in this case tobovine serum albumin (BSA). Because of its single free thiol group oncysteine 34, BSA has been used for in vitro demonstration of adductformation with electrophilic compounds (Satoh et al., Proc. Nat. Acad.Sci. USA 103:768-773, 2006). In order to monitor this reaction, wesynthesized CA-biotin (CAB), in which biotin is conjugated at thecarbonic acid site of CA with a chemical linker. Withperoxidase-conjugated streptavidin as a probe, we could detect adductformation of BSA with CAB. For this purpose, BSA was mixed with vehicleor CAB in PBS at room temperature for 5 h, electrophoresed, and thenprobed with streptavidin. After exposure to CAB, a single band (68 kDa)corresponding to the complex of BSA and CAB was detected. In thismanner, we demonstrated that CAB could form an adduct with BSA in adose-dependent manner.

Additionally, we examined whether cysteine thiol was essential forformation of the complex. We reasoned that if carbon #14 of CA binds tothe free cysteine of BSA, then pretreatment with NEM, an irreversiblethiol alkylating agent, should abolish this binding. We found thatpretreatment with NEM depressed the streptavidin signal in adose-dependent manner, while the total protein remained virtually thesame, as judged from Coomassie brilliant blue-staining of the gel. Theseresults suggest that cysteine thiols are a target of CA.

CA Activates the Keap1/Nrf2/ARE Pathway.

Neuroprotective effects of electrophilic compounds are often manifestvia activation of the Keap1/Nrf2 pathway (Satoh et al., Proc. Nat. Acad.Sci. USA 103:768-773, 2006; Kraft et al., J. Neurosci. 24:1101-1112,2004; Shih et al., J. Neurosci. 10321-10335, 2005). The initial reactionin this activation cascade is the binding of an electrophilic compoundto specific cysteines on Keap1 protein (Hong et al., Chem. Res. Toxicol.18: 1917-1926, 2005; Eggler et al., Proc. Nat. Acad. Sci. USA102:10070-10075, 2005; Zhang et al., Mol. Cell. Biol. 10941-10953,2004). Such binding initiates a cellular transcription pathway leadingto induction of phase 2 enzymes (Talalay, Biofactors 12:5-11, 2000;Padmanabham et al., Mol. Cell 21:689-700, 2006; Itoh et al., Free Radic.Biol. Med. 36:1208-1213, 2004). Therefore, we examined the domains ofKeap1 (designated BTB, IVR, and DGR) to determine those essential forbinding to CA. Accordingly, for co-immunoprecipitation experiments, wetransfected COS7 cells with DNA expressing either HA-tagged wild typeKeap1 protein (HA-WT Keap1) or various deletion mutants of tagged Keap1protein (HA-ΔBTB Keap1, HA-ΔIVR Keap1, and HA-ΔDGR Keap1). Thetransfected COS7 cells were then treated with vehicle or 10 μM CAB andlysed. We quantified the expression level of Keap1 protein in total celllysates with anti-HA antibody. Each Keap1 mutant protein was expressedat a similar level of the predicted molecular weight (57 kDa for HA-ΔBTBKeap1, 53 kDa for HA-ΔIVR Keap1, and 37 kDa for HA-ΔDGR Keap1). Next, inorder to detect Keap1/CAB complexes, we immunoprecipitated cell lysateswith streptavidin and probed with anti-HA antibody. A 73-kDa protein,corresponding to Keap1-WT bound to CAB, was observed in cells treatedwith CAB. ΔDGRKeap1 also manifested strong binding to CAB. In contrast,IVR Keap1 showed very little binding to CAB, whereas ΔBTB Keap1displayed no binding at all. Taken together, these results areconsistent with the notion that maximal CA binding requires the BTB andIVR domains of Keap1 protein (Hosoya et al., J. Biol. Chem.29:27244-27250, 2005; Kobayashi et al., Mol. Cell. Biol. 26:221-229,2006). The requirement of the BTB domain for CA binding is in accordwith prior results (Hong et al., Chem. Res. Toxicol. 18:1917-1926, 2005;Eggler et al., Proc. Nat. Acad. Sci. USA 102:10070-10075, 2005; Zhang etal., Mol. Cell. Biol. 24:10941-10953, 2004).

After binding of the electrophilic quinone-form of CA to Keap1 protein,activation of the Keap1/Nrf2 pathway requires nuclear translocation ofNrf2 (Talalay, Biofactors 12:5-11, 2000; Padmanabham et al., Mol. Cell21:689-700, 2006; Itoh et al., Free Radic. Biol. Med. 36:1208-1213,2004). Thus, we next examined the intracellular distribution of Nrf2protein in COS7 cells by immunofluorescence. Under basal conditions,Nrf2 protein was predominantly localized in the cytoplasm, but wastranslocated into the nucleus upon exposure to 10 μM CA, suggesting thatNrf2 was translocated in response to the binding of CA to Keap1 protein.Similar to COS7 cells, neural PC12h cells transfected with Keap1(non-HA-tagged) expression vector and exposed to CA formed CA/Keap1complexes.

Phase 2 enzymes represent an important effector activated byelectrophilic-induction of the Keap1/Nrf2 pathway. In order to detectinduction of the phase 2 enzymes γ-GCL light chain (GCL-L), γ-GCL heavychain (GCL-H), HO-1, and NQO1, we performed RT-PCR using mRNA of PC12hcells pretreated with 10 μM CA. The cyclophilin A (CYPA) gene was usedas an internal positive control. All of the phase 2 genes were inducedby CA after a 6-24 h incubation. These results are consistent with thenotion that CA induced a set of phase 2 genes, possibly throughactivation of the Keap1/Nrf2 pathway, as previously shown for otherelectrophiles.

Next, we examined the involvement of the Keap1/Nrf2 pathway moreprecisely by studying activation of the ARE. This transcription elementresponds to the Keap1/Nrf2 pathway; we monitored activation of the AREby performing luciferase reporter gene assays using PC12h cellstransfected with ARE(GSTYa)-luciferase (Alam et al., J. Biol. Chem.274:26071-26077, 1999) in the presence or absence of an Nrf2dominant-negative (DN) or Keap1 expression vector. Based on luciferaseactivity, CA (10 μM) stimulated expression of ARE-basedtranscription >10-fold. In contrast, CA-stimulated activation of the AREwas significantly repressed by co-transfection with Nrf2-DN or Keap1.This series of experiments suggests that CA activates the ARE via theKeap1/Nrf2 pathway.

CAB also significantly activated the ARE, but its potency wassubstantially lower than that of CA. For example, 20 μM CAB activatedthe ARE to a much lesser extent than 10 μM CA. Similarly, a greaterlevel of CAB than CA was required to protect PC12h cells againstoxidative glutamate toxicity. Thus, it appears that several times thedose of CAB than CA is required to activate the Keap1/Nrf2 pathway. CAprotects PC12h cells by activating the Keap1/Nrf2 pathway.

Since many phase 2 enzymes are involved in the redox regulation ofcells, induction of these enzymes often affords resistance to oxidativestress (Talalay, Biofactors 12:5-11, 2000; Padmanabham et al., Mol. Cell21:689-700, 2006; Itoh et al., Free Rad. Biol. Med. 36:1208-1213, 2004).Thus, we examined whether CA could protect PC12h cells from suchinsults. We prepared naïve PC12h cells, Nrf2WT-expressing PC12h cells(PC12hW1B), and Nrf2DN-expressing PC12h cells (PC12hD5D). In order toexamine the effects of the Keap1/Nrf2 pathway on cell survival in theface of oxidative stress, we exposed the cells to a high concentrationof glutamate. In PC12h cells, high (millimolar) concentrations ofglutamate induce oxidative cell death primarily by depletingintracellular GSH because of inhibition of cystine influx (Pereira andOliveira, Free Rad. Biol. Med. 23:637-647, 1997). To visualize survivingand dead cells, we stained cultures with fluorescein diacetate (FDA) andpropidium iodide (PI), respectively. Additionally, cell survival wasquantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay (FIG. 4A). We found that glutamate (1-10 mM) induceddeath of PC12h cells within 20 h whereas PC12hW1B cells, overexpressingNrf2WT, were highly resistant to this form of oxidative stress. PC12hD5Dcells, expressing Nrf2DN, exhibited increased susceptibility to celldeath. These results suggest not only that Nrf2 protein can protectcells but that endogenous Nrf2 is involved in the cytoprotectiveresponse against oxidative stress.

Next, we examined activation of the ARE by CA compared to sulforaphane,an electrophilic compound produced by plants and previously shown topotently activate the Keap1/NRf2 pathway (Kraft et al., J. Neurosci.24:1101-1112, 2004; Hong et al., Chem. Res. Toxicol. 18:1917-1926,2005). Surprisingly, on an equimolar basis CA activated the ARE to amuch greater extent than sulforaphane (FIG. 4B). This difference in AREactivation appears to be critical for cell survival because sulforaphanedid not protect PC12h cells against oxidative glutamate toxicity in ourassays. This result is also consistent with the notion that the abilityof CA to activate the ARE coincides with its neuroprotective effect, asobserved below.

We next tested the hypothesis that CA-induced neuroprotection ismediated by the Keap1/NRf2 pathway. We found that 0.1-1 μM CAdose-dependently protected PC12h cells against oxidative glutamatetoxicity (FIG. 4C). However, the protective effects afforded by CA weresignificantly suppressed in PC12hD5D (FIG. 4C). In contrast, PC12hWIBmanifested less cell death after an oxidative glutamate insult. Theseresults suggest that CA protected PC12h cells against oxidative stressin an Nrf2-dependent manner.

CA Protects Cortical Neurons Via Activation of the Keap1/Nrf2 Pathway.

We also assessed whether CA could protect immature cortical neurons inprimary culture from an oxidative glutamate insult; these cells do notyet express functional glutamate/NMDA receptors and consequently succumbto non-receptor-mediated oxidative cell death due to inhibition ofcystine influx, similar to PC12h cells. We found that CA protected thesecortical neurons from exposure to glutamate or rotenone. Glutamate (2mM) and rotenone (300 nM) decreased the number of MAP2 and NeuN-positivecells to 30.8±2.5% and 25.8±1.9% of the control value, respectively. Incontrast, CA (3 μM) increased cell survival in the face of these insultsto 73.8±3.7% and 80.4±3.4%, respectively. These results suggest that CAprotects primary CNS neurons against oxidative stress.

Next, we examined the actions of CA on excitotoxic (NMDAreceptor-mediated) neuronal cell death. In this case, we used moremature cerebrocortical neurons in primary culture that expressed NMDAreceptors. NMDA reduced the number of viable cells to 15±1.6%, whereas 3μM CA increased the survival to 36±2.3%. Taken together, these resultssuggest that CA protected cultured primary neurons from NMDAreceptor-mediated excitotoxicity as well as against non-receptormediated oxidative stress.

To confirm that CA activated the ARE in these cortical cultures, weperformed luciferase reporter gene assays. ARE-mediated transcriptionincreased 2.3-fold in the presence of 3 μM CA, and this effect wasabrogated by Nrf2-DN, suggesting that CA activated the ARE in anNrf2-dependent manner. CA accumulates both in neurons and innon-neuronal cells (FIG. 5).

In order to determine the site(s) of action of CA, we treated mixedneuronal/glial cortical cultures (E17, DIV21) with CAB, and then stainedthem with rhodamine-conjugated streptavidin after fixation.Concurrently, the cultures were stained with antibody against theneuronal marker MAP2 or the non-neuronal cell marker S10013. At lowconcentrations of CAB (e.g., 3 μM), neurons were not strongly labeled,whereas non-neuronal cells were strongly positive for CAB-streptavidin,indicating that CAB had accumulated in non-neuronal cells rather than inneurons. At higher concentrations of CAB (e.g., 10 μM), neurons werealso labeled. These results suggest that CA accumulated in non-neuronalcells at low concentrations but also in neurons at higherconcentrations.

CA Accumulation in the Brain.

For the development of CA as a protective agent againstneurodegenerative diseases, penetration into the brain is an essentialrequirement. To examine this aspect, we administered CA orally to mice(3 mg per 25 g mouse) and measured the level of CA (catechol-form) inserum and brain parenchyma by HPLC. Within 1 h, CA reached significantlevels in the brain, suggesting that CA was able to penetrate theblood-brain barrier.

Next, we examined whether CA exhibited activity in the brain. Mice wereallowed free access to food containing 0.03% CA for a week. Their brainswere then removed, and extracts were prepared and measured for GSH andGSSG levels. Under these conditions, CA increased both total GSH+GSSGand the ratio of GSH/GSSG (FIG. 6). During these experiments, total RNAwas also extracted from the brain and subjected to RT-PCR; we foundsignificant induction of the phase 2 enzymes HO-1 and γ-GCS, consistentwith the notion that CA activates the ARE, thus inducing phase 2 enzymesand increasing GSH levels. Therefore, we concluded thatorally-administered CA could not only reach the brain but was alsoeffective in activating potentially neuroprotective pathways. Theseexperiments also suggest that chemical reactions occur in vivo thatconvert CA, which is normally not an electrophile in its catechol or“pro-drug” form,” to an electrophilic (quinone) compound, because CAadministration resulted in induction of phase 2 enzymes in the brain.

Most importantly, the elucidation of this mechanism set the stage forwork with this electrophilic precursor compound to test itsneuroprotective efficacy in vivo.

CA Protects the Brain from MCAO/Reperfusion Injury In Vivo.

Next, we tested whether CA could decrease the volume of cerebralinfarcts after MCAO/reperfusion injury. CA or vehicle (10% DMSO in PBS)was injected intraperitoneally 1 h prior to MCAO. The volume of braininfarction (corrected for possible edema) was assessed by TTC stainingof coronal sections 24 h after the onset of reperfusion. MCAO inducedsevere brain damage in vehicle-injected mice; the infarct volume was51.2±2.4% of the ipsilateral hemisphere, similar to prior reports (Gu etal., Science 297:1186-1190, 2002; Gu et al., J. Neurosci. 25:6401-6408,2005; Satoh et al., Proc. Nat. Acad. Sci. USA 103:768-773, 2006). Incontrast, CA significantly reduced the infarct volume (to 34.5±3.6%)(FIG. 7), consistent with the notion that CA was neuroprotective invivo.

Discussion

In the present experiments, we found that CA is neuroprotective both invitro and in vivo from glutamate/oxidative stress and cerebral ischemia.Concerning the chemical entity that affords this protection, thequestion arises if the real effector is the catechol- or quinone-type ofCA? If the catechol-type CA is the effector, it must protect neuronsthrough its antioxidant activity. In contrast, if the quinone-type isthe effector, it would protect neurons by activating the Keap1/Nrf2pathway. In the present study, we found that the effector is thequinone-type and not the catechol-type molecule. This conclusion isbased on the following results: (1) Quinone-type CA, but notcatechol-type, activated the Keap1/Nrf2 pathway; (2) Activation of theKeap1/Nrf2 pathway by stable expression of WT Nrf2 protected PC12h cellsagainst oxidative stress; (3) Inhibition of the Keap1/Nrf2 pathway bystable expression of DN Nrf2 increased oxidative stress-induced celldeath and reduced protection by CA. Thus, although catechol-type CAcould potentially exert antioxidant activity by donating a pair ofelectrons to oxygen radicals, this mechanism appears to play a limitedrole in the neuroprotection observed here.

The time course of conversion from catechol to quinone CA was ratherslow in the cell-free system that we used. Then, how can the quinoneform of CA be neuroprotective? One plausible explanation lies in thedifference between the cell-free and cell-culture experiments employedhere. Since cells have many thiols, for example, on GSH andcysteine-bearing proteins, quinones react rapidly with these thiols toform an adduct; removal of the quinone-type of CA by adduct formationshifts the equilibrium between free catechol and free quinone towardsthe quinone. Thus, conversion should be much faster in cell-based thanin cell-free systems. Additionally, there are differences in thiolreactivity between GSH and various cysteine residues in proteins. Forexample, some proteins have potentially reactive cysteines, which can beeasily converted to the thiolate anion if they are located in a motif ofbasic amino acids. Cysteine (151) of Keap1 is a typical example of suchan active cysteine. This cysteine appears to be a sensor forelectrophilic compounds. Thus, cysteine (151) of Keap1 should be morereactive with electrophilic compounds than GSH. Thus, the sustainedpresence of low concentrations of quinone-type CA may preferentiallyreact with Keap1 rather than with GSH, and may thereby contribute toactivation of the cell defense system. We speculate that this may be oneof the reasons that CA is much less toxic than NEPP11, which itself isan electrophilic compound.

In the present series of in vitro experiments, we showed the chemicaland molecular mechanisms whereby CA protected neurons against oxidativestress. CA is converted from an electrophilic precursor (orpro-electrophilic) compound to an electrophilic form, thereby activatingthe neuroprotective Keap1/Nrf2 pathway (FIG. 8). The chemical mechanisminvolves a catechol-type CA that is oxidized to a quinone-type, with thecarbon at position 14 [C(14)] becoming electrophilic. This quinone-typeCA is subject to nucleophilic attack by the cysteine thiol of GSH orvarious other proteins to form an adduct. Importantly, via this chemicalreaction, the cysteine thiol of Keap1 protein forms a Keap1-CA adduct,resulting in release of Nrf2 protein from the Keap1/Nrf2 complex. Nrf2can then be translocated into the nucleus, where it activatestranscription of phase 2 enzymes via ARE transcriptional elements. Thesephase 2 enzymes improve the redox state of neurons, contributing to theanti-oxidant defense system.

Although electrophilic compounds can possess a variety of chemicalstructures, the enone-type (e.g., NEPP11) and catechol-type (e.g., CAand TBHQ) represent major groups. From our prior findings and those ofother groups (Satoh et al., Proc. Nat. Acad. Sci. USA 103:768-773, 2006;Kraft et al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.25:10321-10335, 2005), we have proposed that the cellular distributionin the brain of enone- and quinine-type electrophilic compounds may bedifferent. Enone-type electrophiles, including dienones such as NEPP11,appear to act preferentially in neurons based on our earlier findingsthat NEPP11 accumulates in neurons as opposed to astrocytes, andconsequently induces neuronal HO-1 (Satoh et al., Proc. Nat. Acad. Sci.USA 103:768-773, 2006). In contrast, catechol-type electrophiles,including TBHQ, preferentially act on astrocytes, as evidenced by thefact that TBHQ activates the ARE in astrocytes and not in neurons (Kraftet al., J. Neurosci. 24:1101-1112, 2004; Shih et al., J. Neurosci.25:10321-10335, 2005). In light of these findings, we proposed that theenone-type electrophile NEPP11 exerts a direct neuroprotective effectwhile the catechol-type TBHQ exerts a paracrine-type of neuroprotectiveaction (Satoh and Lipton, Trends Neurosci. 30:38-45, 2007).Interestingly, CA appears to afford both direct and paracrine (hence,“mixed”) neuroprotective effects, as discussed below.

In the immunohistochemical experiments we conducted using CAB, thislabeled form of CA accumulated in non-neuronal cells at lowconcentrations (3 μM) and in neurons at higher concentrations (10 μM).However, ARE activation and consequent neuroprotection afforded by CABwere much weaker than by CA. This difference may have been due to lowerpermeability of cell membranes to CAB or lower binding affinity of CABfor Keap1 protein. In light of this difference, we estimate that <10 μMCA accumulates in both non-neuronal and neuronal cells. Thus, it isreasonable to propose that CA exerts actions on both cell types, similarto the astrocyte-mediated neuroprotective action previously seen forTBHQ and the neuronal-mediated neuroprotection previously observed forNEPP1.

An important advantage of electrophiles such as NEPP11 and CA asneuroprotective compounds is their transcriptional activation ofantioxidant phase 2 enzymes. This type of neuroprotection could be ofpotential benefit in chronic neurodegenerative diseases such asParkinson's and Alzheimer's diseases. Previously, NEPP11 was shown toprotect neurons via electrophilic chemical reaction (Satoh et al., Proc.Nat. Acad. Sci. USA 103:768-773, 2006; Satoh and Lipton, TrendsNeurosci. 30:38-45, 2007). However, NEPP11 has a serious problem as apotential therapeutic agent because systemic administration can resultin reaction with thiol substrates prior to reaching the intended targetin the brain. It would be far better to have a pro-electrophiliccompound that remains non-reactive until it is converted to anelectrophile by the oxidative insult at the pathological site of itsintended action. Such as drug would represent what has been termed a“pathologically-activated therapeutic” (Lipton, Nature 428:473, 2004),and we believe that CA may constitute such an agent. We feel that CA hastwo clear-cut advantages over NEPP11 and similar compounds: (i) lowpotential toxicity due to conversion of the pro-drug to an electrophileby the oxidative insult at the pathological site, and (ii) effectivepenetrance into brain tissue. These characteristics emanate from thechemical structure of CA. Accordingly, when the catechol is oxidized toa quinone, it becomes more hydrophobic and will tend to stay in theinjured tissue.

Moreover, CA is less toxic than NEPP11 in vitro. CA manifests a largetherapeutic index; i.e., 100 μM CA is not toxic to neural cells, whereasas little as 1-3 is neuroprotective. The lower toxicity of CA may alsobe accounted for by the fact that, unlike NEP11, CA only becomeselectrophilic at or near the site of injury. Oxidative stress plays acritical role in the progression of neurodegenerative disorders (Coyleand Puttfarcken, Science 262:689-695, 1993). We demonstrate here thatthis pathological level of oxidation can be used to activatepro-electrophilic compounds at the target site to provideneuroprotection where it is needed. Thus, this approach represents anovel strategy against neurodegenerative disorders by activatingneuroprotective pro-electrophilic drugs via the very pathologicalactivity that they are meant to combat. Moreover, to our knowledge, thisstudy is the first demonstration of a natural product protecting neuronsby thiol (S-) alkylation, resulting in activation of the Keap1/Nrf2pathway. Thus, naturally-occurring pro-electrophilic compounds producedby plants are candidate neuroprotective agents for the treatment ofneurodegenerative diseases.

REFERENCES

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. A compound, or pharmaceutically acceptable prodrug, salt or solvatethereof, of Formula IV:

wherein: X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₇ are each independently H, OH,oxo, or Y; Y is B—C-D or C—B-D or C—B—C-D, any of which may be attachedto a ring carbon to form a fused ring; B is selected from the groupconsisting of null, carbonyl, carboxy, ether, sulfanyl, amino, —NHC(O)—and —C(O)NH—, any of which is optionally substituted; C is selected fromthe group consisting of null, alkyl, cycloalkyl, alkenyl, cycloalkenyl,aryl, arylalkyl, and arylalkenyl, any of which is optionallysubstituted, and which may be attached to a ring carbon so as to form afused ring; and D is selected from the group consisting of null,carboxy, benzoic acid, hydroxybenzoic acid, SO₃H, PO₃, NO₃, NO₂, NO,amino, hydroxyl; wherein X₂₆ is methyl; and wherein B, and C areoptionally substituted with one or more substituents independentlyselected from lower alkyl, lower alkenyl, lower alkenyl, lower alkanoyl,lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lowerhaloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy,lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy,oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lowercarboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy,amino, lower alkylamino, arylamino, amido, nitro, thiol, loweralkylthio, arylthio, lower alkylsulfonyl, lower alkylsulfonyl,arylsulfonyl, arylsulfonyl, arylthio, sulfonate, sulfonic acid,trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl,thiophene, furanyl, lower carbamate, and lower urea.
 2. The compound ofclaim 1 wherein C is an alkyl.
 3. The compound of claim 1 wherein X₂₇and X₂₄ are each independently methyl.
 4. The compound of claim 3wherein X₂₁, X₂₂, X₂₃ and X₂₅ are each null. 5-76. (canceled)